Bacterial cellulose based &#39;green&#39; composites

ABSTRACT

‘Green’ composites are fabricated using resins, such as soy-based resins, and reinforced with crystalline high strength bacterial cellulose (BC) fibers. Bacterial cellulose is produced by providing a bacterial cellulose-producing bacterium such as  Acetobacter xylinum ; providing an inexpensive bacteria nutritional medium; culturing the bacterium in the bacteria nutritional medium under conditions to produce bacterial cellulose; and isolating bacterial cellulose produced by cultured bacteria from the bacteria nutritional medium. The bacteria nutritional medium comprises an inexpensive carbon source that is a plant-based seed extract. The seed extract is derived from a plant-based seed comprising soluble sugars.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of co-pending U.S.provisional patent application Ser. No. 61/179,257, entitled BacterialCellulose Based ‘Green’ Composites, filed May 18, 2009, which isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The disclosed invention was made with government support under contractno. ITA-08-07400 from the Department of Commerce (National TextileCenter). The government has rights in this invention.

1. TECHNICAL FIELD

The invention relates in general to bio-based materials and specificallyto bacterial cellulose (BC) and BC based composites. The invention alsorelates to methods for producing bacterial cellulose (BC) basedcomposites.

2. BACKGROUND OF THE INVENTION

During past several decades, new advanced composites with excellentmechanical properties have been developed and used as metal replacement.However, most composites are made using synthetic non-degradable fibers,such as carbon, aramid and glass and polymers (resins), such aspolyetheretherketone (PEEK) and epoxy. They cannot be recycled or reusedeasily and most end up in landfills. These composites pose a serioussolid waste disposal problem due to decreasing landfill space,widespread litter, and pollution of marine environments.

Bacterial cellulose (BC) produced by Acetobacter xylinum, is a promisingsustainable and biodegradable fibrous material and has the same chemicalstructure as the plant-based cellulose. However, BC fibers havediameters in the range of a few nano-meters and display many uniqueproperties including higher purity, higher crystallinity, higher degreeof polymerization, higher tensile strength, higher modulus and strongbiological adaptability (Iguchi, M.; Yamanaka, S.; Budhiono, A. (2000).Bacterial cellulose—a masterpiece of Nature's arts. Journal of MaterialsScience, 35 (2), 261-270; Baeckdahl, H.; Helenius, G.; Bodin, A.;Nannmark, U.; Johansson, B. R.; Risberg, B.; Gatenholm, P. (2006).Mechanical properties of bacterial cellulose and interactions withsmooth muscle cells. Biomaterials, 27 (9), 2141-2149; Klemm, D.;Schumann, D.; Udhardt, U.; Marsch, S. (2001). Bacterial synthesizedcellulose—artificial blood vessels for microsurgery. Progress in PolymerScience, 26(9), 1561-1603; Klemm, D.; Heublein, B.; Fink, H. P.; Bohn,Andreas. (2005). Cellulose: Fascinating biopolymer and sustainable rawmaterial. Angewandte Chemie, International Edition, 44(22), 3358-3393;Fink, H. P.; Weigel, P.; Purz, H. J.; Ganster, J. (2001). Structureformation of regenerated cellulose materials from NMMO-solutions.Progress in Polymer Science, 26(9), 1473-1524). The BC material has beenused in a variety of applications including artificial skin and bloodvessel, binding agent, loud speaker diaphragms, paper, foods, textile,composite membranes, etc. (Wan et al., 2006; Fontana, J. D.; De Souza,A. M.; Fontana, C. K.; Torriani, I. L.; Moreschi, J. C.; Gallotti, B.J.; De Souza, S. J.; Narcisco, G. P.; Bichara, J. A.; Farah, L. F. X.(1990). Acetobacter cellulose pellicle as a temporary skin substitute.Applied Biochemistry and Biotechnology, 24-25, 253-264; Shibazaki, H.;Kuga, S.; Onabe, F.; Usuda, M. (1993). Bacterial cellulose membrane asseparation medium. Journal of Applied Polymer Science, 50 (6), 965-969;Svensson, A.; Nicklasson, E.; Harrah, T.; Panilaitis, B.; Kaplan, D. L.;Brittberg, M.; Gatenholm, P. (2005) Bacterial cellulose as a potentialscaffold for tissue engineering of cartilage. Biomaterials, 26 (4),419-431). Many pure sugars have been used as carbon source for BCculture. Among them mannitol and fructose are the most common and haveshown excellent results in terms of BC production (Hong, F.; Qiu, K.(2008). An alternative carbon source from konjac powder for enhancingproduction of bacterial cellulose in static cultures by a model strainAcetobacter aceti subsp. xylinus ATCC 23770. Carbohydrate Polymers, 72(3), 545-549). However, cost of these sugars is high and as a result,they are not considered to be ideal for large scale BC production. As aresult, many attempts have been made to obtain higher BC yields as wellas to reduce the cost of the carbon sources with some success. Theseinclude konjac powder hydrolyzate (Hong, F.; Qiu, K. (2008). Analternative carbon source from konjac powder for enhancing production ofbacterial cellulose in static cultures by a model strain Acetobacteraceti subsp. xylinus ATCC 23770. Carbohydrate Polymers, 72 (3),545-549), sugarcane molasses (Keshk, S.; Sameshima, K. (2006). Theutilization of sugar cane molasses with/without the presence oflignosulfonate for the production of bacterial cellulose. AppliedMicrobiology and Biotechnology, 72 (2), 291-296), beet molasses (Keshk,S.; Razek, T. Sameshima, K. (2006). Bacterial cellulose production frombeet molasses. African Journal of Biotechnology, 5(17), 1519-1523) andprocessed rice bark (Goelzer, F. D. E.; Faria-Tischer, P. C. S.;Vitorino, J. C.; Sierakowski, Maria-R.; Tischer, C. A. Production andcharacterization of nanospheres of bacterial cellulose from Acetobacterxylinum from processed rice bark. (2009). Materials Science andEngineering, C: Materials for Biological Applications, 29(2), 546-551).While some of these sources may be used for industrial BC production inthe near future, there is significant scope to further lower the cost ofBC production and expand its use in many mass volume applications.

Defatted soy flour (SF) is obtained after extracting oil from thesoybeans. It consists mainly of protein (52-54%), sugars (30-32%),dietary fiber (2-3%), minerals and ash (3-6%) and moisture (6-8%). Thesoybean is a legume species native to East Asia and is classified as anoilseed. It is an annual and economic crop and has been abundantlyproduced and used in some countries for over 5,000 years (Endres J. G.(2001). Soy protein products: Characteristics, nutritional aspects andutilization, revised and expanded edition. AOCS Press, pp. 4-18).Currently, it is an important global crop and provides major amount ofedible oil and protein (Martin, H.; Laswai, H.; Kulwa, K. (2010).Nutrient content and acceptability of soybean based complementary food.African Journal of Food, Agriculture, Nutrition and Development, 10(1),2040-2049). The soybean has been shown to contain decent amount ofsugars, including fructose, glucose, sucrose, raffinose and stachyose(Giannoccaro, E.; Wang, Y. J.; Chen, P. (2008). Comparison of two HPLCsystems and an enzymatic method for quantification of soybean sugars.Food Chemistry, 106, 324-330). Fructose, glucose and sucrose have beenused as routine carbon sources for BC production in previous reports(Yang et al., 1997). It has also been reported that raffinose andstachyose could be metabolized by lactic acid bacteria (Wang, Y. C.; Yu,R. C.; Yang, H. Y.; Chou, C. C. (2003). Sugar and acid contents insoymilk fermented with lactic acid bacteria alone or simultaneously withbifidobacteria. Food Microbiology, 20(3), 333-338). However, BC has notbeen produced using the soy flour extract (SFE), an inexpensiveby-product of SF, as a carbon source by Acetobacter xylinum.

There is therefore a need in the art for advanced composites withexcellent mechanical properties to use as metal replacements that aresustainable, biodegradable and inexpensive to produce. There is furthera need in the art for inexpensive industrial BC production.

3. SUMMARY OF THE INVENTION

A method for producing bacterial cellulose (BC) is provided comprising:

providing a bacterium wherein the bacterium is a bacterialcellulose-producing bacterium;providing a bacteria nutritional medium;culturing the bacterium in the bacteria nutritional medium underconditions to produce BC; andisolating BC produced by cultured bacteria from the bacteria nutritionalmedium,wherein:the bacteria nutritional medium comprises a carbon source,the carbon source is a plant-based seed extract, andthe plant-based seed extract is derived from a plant-based seedcomprising soluble sugars.

Soluble sugars suitable for use in the methods of the invention caninclude, but are not limited to: fructose, glucose, sucrose, raffinose,stachyose, galactose and maltose. Any seed comprising soluble sugars canbe used for the seed extract. Seeds comprising soluble sugars are knownin the art.

In one embodiment, the carbon/sugar source is an inexpensive and orsustainable source such as soy flour extract (SFE).

In another embodiment, the bacteria is Acetobacter xylinum.

In another embodiment, the e seed is soy, wheat, corn or a legume.

In another embodiment, the seed extract is soy flour extract (SFE).

In another embodiment, the step of isolating BC comprises harvesting BCpellicles produced on the surface of the bacteria nutritional medium.

In another embodiment, the bacteria nutritional medium comprisesmicrofibrillated cellulose (MFC), nanofibrillated cellulose (NFC),nanoparticles, nanoclay or nanocubes.

In another embodiment, the bacteria nutritional medium comprises fibers.

In another embodiment, the fibers are transparent.

In another embodiment, the fibers comprise a natural cellulose-based orprotein-based material.

In another embodiment, the natural cellulose-based material is selectedfrom the group consisting of cotton, linen, flax, sisal, ramie, hemp,kenaf, jute, bamboo, banana, pineapple, kapok and cellulose andcombinations thereof.

In another embodiment, the natural protein-based material is selectedfrom the group consisting of wool, silk, angora, cashmere, mohair,alpaca, milk protein and soy protein and combinations thereof.

In another embodiment, the fibers comprise a polymeric material.

In another embodiment, the polymeric material is cellulose acetate,nylon, rayon, modacrylic, olefin, acrylic, polyester, polylactic acid,polylactic-co-glycolic acid (PLGA), polyurethane, aramid (e.g. KEVLAR®),or ultrahigh molecular weight polyethylene, (e.g. SPECTRA® or DYNEEMA®).

In another embodiment, the fibers comprise carbon (e.g., carbon fiber)or glass (e.g., fiberglass). In another embodiment, the fiber isintroduced into the nutritional medium before or during the culturingstep.

In another embodiment, the method comprises drying or hot-pressing theisolated BC, thereby forming a membrane.

In another embodiment, the method comprises immersing or soaking theisolated BC in a resin.

In another embodiment, the method comprises crosslinking BC and theresin with a crosslinking agent.

In another embodiment, the crosslinking agent is glutaraldehyde (GA),glyoxal, rutin, quercetin, a hydroxyl, a diol or ethylene glycol.

A method for producing a soy flour extract (SFE) for use in theproduction of bacterial cellulose (BC) is also provided. In oneembodiment, the method comprises providing soy flour (e.g., in apowder), preparing a soy flour mixture by mixing the soy flour withwater (e.g., at a ratio of 3 parts soy flour:17 parts water); adjustingthe pH value of the soy flour mixture to a desired pH (e.g., to pH 4.5by adding, e.g., hydrochloric acid); heating the soy flour mixture; andfiltering the soy flour mixture to remove solid contents (e.g.,insoluble protein); evaporating the filtrate to obtain a SFE with adesired sugar concentration.

A method for producing a soy flour extract (SFE) for use in theproduction of bacterial cellulose (BC) is also provided. The methodpreferably comprises the step of autoclaving a soy flour extract (SFE).A soy flour extract (SFE) is also provided, for use in the production ofbacterial cellulose (BC) and that is enriched for fructose and glucose.

A composition is also provided comprising bacterial cellulose (BC) and asoy-based resin. In one embodiment, the composition comprises 20 to 60%BC by weight.

In another embodiment, the composition is crosslinked.

A composition is also provided comprising bacterial cellulose (BC); andan agent selected from the group consisting of microfibrillatedcellulose (MFC), nanofibrillated cellulose (NFC), cellulose nanowhisker,nanoparticle, nanoclay or nanocube, wherein the agent is interwoven orintercalated with the BC. In one embodiment, the composition comprises aresin.

In another embodiment, the resin is transparent.

In another embodiment, the resin is selected from the group consistingof natural resin, plant-based resin and non-toxic resin.

In another embodiment, the resin is biodegradable.

In another embodiment, the resin is water soluble.

In another embodiment, the natural or plant-based resin is a soy-basedresin.

In another embodiment, the resin is a petroleum-based resin.

In another embodiment, the petroleum-based resin is an epoxy, vinyl, orunsaturated polyester-based resin.

In another embodiment, the resin is polyethylene oxide (PEO).

In another embodiment, the resin is polyvinyl alcohol (PVA).

In another embodiment, the resin is polyhydroxy alkanoate (PHA).

In another embodiment, the composition is a membrane.

In another embodiment, the composition comprises fibers.

In another embodiment, the fibers are transparent.

In another embodiment, the fibers comprise a natural cellulose-based orprotein-based material.

In another embodiment, the natural cellulose-based material is selectedfrom the group consisting of cotton, linen, flax, sisal, ramie, hemp,kenaf, jute, bamboo, banana, pineapple, kapok, and combinations thereof.Any other natural cellulose fibers known in the art can also be used.

In another embodiment, the natural protein-based material is selectedfrom the group consisting of wool, silk, angora, cashmere, mohair,alpaca, milk protein, spider silk, and soy protein and combinationsthereof.

In another embodiment, the fibers comprise a polymeric material. Inanother embodiment, the polymeric material is cellulose acetate, nylon,rayon, modacrylic, olefin, acrylic, polyester, polylactic acid,polylactic-co-glycolic acid (PLGA), polyurethane, aramid (e.g. KEVLAR®),or ultrahigh molecular weight polyethylene, (e.g. SPECTRA® or DYNEEMA®).In another embodiment, the fibers comprise carbon (e.g., carbon fiber)or glass (e.g., fiberglass).

4. BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is described herein with reference to theaccompanying drawings, in which similar reference characters denotesimilar elements throughout the several views. It is to be understoodthat in some instances, various aspects of the invention may be shownexaggerated or enlarged to facilitate an understanding of the invention.

FIG. 1. Consumption of sugars in SFE medium during BC culture byAcetobacter xylinum ATCC 23769. Symbols: ♦, fructose and glucose; ▪,sucrose; ▴, raffinose; x, stachyose.

FIG. 2. BC yield in SFE medium during culture.

FIG. 3. BC yields obtained for different carbon sources.

FIG. 4. BC-SPI (left) and GA treated BC-SPI (right) composites.

FIG. 5. BC-MFC pellicle (left), SEM images of its surface (middle) andcross section (right).

FIG. 6. Tensile properties of BC-soy resin (SPI) composites withdifferent BC contents.

FIG. 7. FTIR spectra of BC, BC-SPI, BC-MFC and BC-MFC-SPI composites (a:BC; b: BC:SPI=1:1; c: BC-MFC; d: BC-MFC:SPI=1:1).

FIGS. 8 a-b. TGA of BC and BC-soy resin (SPI) composite. a: BC; b:BC-soy resin (SPI) (1:1).

FIG. 9. Schematic diagram of microbond test.

FIG. 10. BC-modified sisal fibers in culture media (left) andmicroscopic image of a single BC-modified sisal fiber (right).

FIG. 11. IFSS of sisal fiber-SPI and BC coated sisal fiber-SPI.

FIG. 12. BC-Sisal-soy resin composites.

FIG. 13. Example of a silicon mold and its application for alignedBC-Sisal and BC-Sisal-Resin production.

FIG. 14. BC-PVA (left) and BC-PEO (right) composite films.

FIGS. 15 a-f. SEM images of BC and BC-PEO composite. a: BC surfacemorphology. b: BC morphology. c: BC-PVA morphology. d: BC-PVAmorphology. e: BC-PEO morphology. f: BC-PEO morphology.

FIG. 16. FTIR spectra of BC, BC-PVA composites and PVA. a: BC; b:BC:PVA=2:1. c: BC:PVA=1:1. d: BC:PVA=1:2. e: PVA.

FIG. 17. FTIR spectra of BC, BC-PEO composites and PEO. a: BC. b:BC:PEO=2:1. c: BC:PEO=1:1. d: BC:PEO=1:2. e: PEO.

FIGS. 18 a-c. TGA of BC, BC-PVA and BC-PEO composites. a: BC; b: BC-PVA(1:1); c: BC-PEO (1:1).

5. DETAILED DESCRIPTION OF THE INVENTION

Methods are provided for fabricating ‘green’ composites using resinsreinforced with crystalline high strength cellulose fibers. In aspecific embodiment, the resin is soy resin. Bacterial cellulose (BC) isa promising biodegradable material with broad potential for compositereinforcement. It is a type of specific cellulose that can be producedby Acetobacter xylinum, a Gram-negative, obligately aerobic bacterium,by culturing Acetobacter xylinum in a nutritional fermentation medium(e.g., at 30° C.) for several days.

In certain embodiments, the nutritional fermentation medium at leastcontains carbon sources (mannitol, sucrose, fructose, etc.) and nitrogensources (peptone, tryptone, yeast extract, etc.), and its optimum pH is5.0.

BC has the same chemical structure as other plant-based cellulose.However, BC is made of fiber diameters of only few nanometers anddisplays many unique properties including higher purity, highercrystallinity, higher degree of polymerization, higher tensile strength(200-300 MPa), higher modulus (up to 78 GPa) and stronger biologicaladaptability. BC material is known in the art for use in a variety ofapplications, including artificial skin and blood vessel, binding agent,loud speaker diaphragms, paper, foods, textile, composite membranes,etc. However, low production rate and high cost of carbon sources havebecome a bottleneck to BC's industrial production and large scale use.

Methods for using inexpensive carbon sources to produce BC are providedthat can be used to reduce the production costs of BC.

BC based ‘green’ and environment-friendly nano-composites are alsoprovided.

In certain embodiments, additional constituents can be added to thebacteria nutritional medium before or during BC synthesis to furtherimprove the mechanical or properties of the synthesized BC.Micro-fibrillated cellulose (MFC) and/or nano-fibrillated cellulose(NFC) can be added to the bacteria nutritional medium to further improvethe mechanical properties of BC. The bacteria excrete a continuousstrand of BC as the bacteria take a random walk among the MFC and/orNFC, resulting in an interpenetrating network of BC, MFC and/or NFC. Inother embodiments, nanoparticles, nanoclay, nanocubes (e.g., halloysite,aluminosilicate nanotube), cellulose nanowhiskers, etc. can be can beadded to the bacteria nutritional medium to improve the mechanical orthermal properties of the synthesized BC. In certain embodiments, theadded fibers, nanoparticles, etc. can be transparent. The resultingmembrane produced from the BC with the added constituents can haveproperties similar to a BC membrane alone. However, in certainembodiments, it can be thicker than the BC membrane alone.

In other embodiments, ‘green’ (e.g., non-toxic, biodegradable),sustainable (derived from renewable resources), or water soluble resinscan be added to reduce composite fabrication to a one-step process.Natural fibers such as sisal and ramie can also be added to form hybrid‘green’ composites with attractive properties.

BC has several advantages over plant cellulose including: finerstructure, no hemicellulose or lignin need to be removed, longer fiberlength and greater strength, can be grown to virtually any shape, andcan be produced on a variety of substrates

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections set forthbelow.

5.1 Bacteria and Culture Media for BC Production

Methods are provided herein for producing bacterial cellulose (BC). Inone embodiment, the method comprises providing a bacterium wherein thebacterium is a bacterial cellulose-producing bacterium; providing abacteria nutritional medium; culturing the bacterium in the bacterianutritional medium under conditions to produce BC; and isolating BCproduced by cultured bacteria from the bacteria nutritional medium,wherein: the bacteria nutritional medium comprises a carbon source, thecarbon source is a plant-based seed extract, and the plant-based seedextract is derived from a plant-based seed comprising soluble sugars.

Many strains of bacteria that synthesize cellulose can be used tosynthesize BC. In a preferred embodiment, Acetobacter xylinum, ATCC23769, can be used (American Type Culture Collection (ATCC), Manassas,Va.).

The bacteria can be maintained using standard culture conditions on agarplates.

Any culture medium known in the art to support the selected bacteria canbe used. For example, SFE medium used for BC production can consist of 5g/L yeast extract, 5 g/L tryptone and autoclaved SFE as the sole carbonsource.

Culture media that can be used can consist of 5-50 g/L of one or morecarbon sources (e.g., stachyose, raffinose, glucose, sucrose, fructose,mannitol, galactose, maltose), 5 g/L yeast extract and 5 g/L tryptone.In one embodiment, 25 g/L is used. Any small sugar known in the art canalso be used.

Bacteria can be maintained using standard culture methods, such as onagar plates containing 25 g/L D-mannitol, 5 g/L yeast extract and 5 g/Ltryptone and 20 g/L agar. The mannitol culture medium used for BCproduction can consist of, for example, 25 g/L D-mannitol, 5 g/L yeastextract and 5 g/L tryptone.

5.2 Bacterial Cellulose (BC) Pellicle Production

The step of culturing the bacterium in the bacteria nutritional mediumunder conditions to produce BC can comprise the step of producing a BCpellicle. To produce the BC pellicle, the bacterial strain is used toinoculate a bacteria nutritional medium (culture medium) as the seedculture. The initial pH value of the medium is adjusted, e.g., to avalue in the range of pH 5.0-6.0.

The pH of the medium can be unregulated during culture/fermentation andmay vary. Changes in pH values may vary depending on the type of sugarin the bacterial nutritional medium (e.g., for mannitol, the pH valueranges from about pH 5.0-6.0 during culture). Some sugars will increasepH of the medium, whereas others will lower pH.

The seed culture is incubated using standard incubation methods (e.g.,at 30° C. and 130 rpm on a rotary shaker for 2 days), and an aliquot ofthis is used to inoculate a desired volume of culture medium in aculture or fermentation vessel for production of BC. The cultivation canbe carried out under standard culture conditions (e.g., at pH 5.0 and30° C. in a static incubator for 10 days). After incubation, the BCpellicle that is produced on the surface of the sugar (e.g., mannitol)culture medium is harvested and washed successively with water and 1%(w/v), aqueous NaOH at 90° C. for 15 min, and then washed by deionizedwater to remove all microbial product contaminants.

In one embodiment, the following sequence of preparation steps can beemployed:

Prepare mannitol culture medium used for BC production which consists of25 g/L D-mannitol, 5 g/L yeast extract and 5 g/L tryptone.

Adjust initial pH value of the medium to 5.0 by acetic acid

Sterilize mannitol culture medium in sterilizer at 121° C. and 15 psifor 15-25 min

Inoculate strain from the agar plate into a conical flask containingmannitol culture medium as the seed culture.

Culture Acetobacter xylinum for seed liquid at 30° C. and 130 rpm on arotary shaker for 2 days.

Transfer 6 mL of seed liquid into a 100-mL culture medium in 500-mLconical flask for production of BC (9 mL of seed liquid into a 150-mLculture medium).

Culture Acetobacter xylinum for BC production at initial pH 5.0 and 30°C. in a static incubator for 10 days.

Harvest BC crude pellicle produced on the surface of mannitol culturemedium.

Wash BC crude pellicle successively with water and 1% (w/v), aqueousNaOH at 90° C. for 15 min, and then remove all microbial productcontaminants by deionized water (BC wet pellicle).

Remove water from the BC either by air dry, oven dry or freeze dry (forBC film).

5.3 Carbon Sources for Bacterial Cellulose (BC) Production

In one embodiment, soy flour extract (SFE), which is derived from soyflour (SF), can be used as the carbon source for BC production. Soyflour extract (SFE) consists of several sugars such as fructose,glucose, sucrose, raffinose and stachyose. Any of these sugars can beused as carbon source for bacterial cellulose production.

Fructose and glucose are preferred carbon sources for BC production ascompared with sucrose, raffinose and stachyose, if the pH value of themedia is kept constant. After autoclaving, the fructose and glucoseconcentration in the SFE medium can increase several-fold and can reach,for example, 7.54 g/L from an initial concentration of 1.92 g/L. Theconcentration of three sugars (fructose, glucose, and sucrose) in theSFE medium that Acetobacter xylinum mainly consumes during the 10-dayculture can be around 25 g/L.

Other sugars known in the art can also be used for production ofbacterial cellulose (BC) using Acetobacter xylinum, e.g., maltose orgalactose.

In a preferred embodiment, the carbon source, e.g., SFE, is autoclavedusing standard methods known in the art. The autoclaving process splitsthe sucrose in the SFE to fructose and glucose. Autoclaving causes asignificant decrease in sucrose concentration and an almost equivalentincrease in the fructose and glucose concentration after autoclaving. Asa result, there can be no change in the combined concentration offructose, glucose and sucrose which preferably remains in the range of23-25 g/L, before and after autoclaving. Autoclaving can be performed onthe carbon source (e.g., SFE) at any suitable point known in the art,e.g., after filtering the carbon source, after it is dried, just beforeusing it, etc. Autoclaving can be performed for 10-40 min, preferablyfor 25 min (an industry norm). Longer times may allow more sucrose tohydrolyze to glucose and fructose. It may also degrade raffinose andstachyose to lower sugars. Many commercially available autoclaves arebuilt for temperature of 121° C. and pressure of 15 psi. Although incertain embodiments these temperature and pressure conditions arepreferred, other suitable temperature and pressure conditions forautoclaving can be easily determined by one of skill in the art. Incertain embodiments, higher temperatures will produce faster sugardegradation.

5.4 Treatment of Soy Flour Powder to Produce SFE

The method disclosed herein for SFE production is extremely inexpensive,convenient and has a high yield.

SFE can be used as an inexpensive carbon/sugar source for the bacterianutritional medium. In one embodiment, SFE is produced by mixing soyflour (7B) powder with deionized water to produce soy flour extract(SFE). Soy flour powder can be initially soaked in water (e.g.,deionized water) in a ratio of around 3:17. The pH value of the mixturecan be adjusted to 4.3-4.7, preferably 4.5, using standard methods,e.g., by adding hydrochloric acid.

The mixture can be incubated, e.g., maintained at 50° C. in a water bathfor 1 hr. After that, the mixture can be filtered to remove the solidcontents (e.g., insoluble protein). The filtrate can be evaporated ordried to obtain the desired suitable main sugar concentration of the SFE(based only on the combined concentration of fructose, glucose andsucrose) which is around 25 g/L for BC culture. As described above,autoclaving can be performed on the SFE at any suitable point known inthe art, e.g., after filtering the carbon source, after it is dried,just before using it, etc.

In other embodiment, sugars for use in the bacteria nutritional mediumcan be extracted from soy protein isolate (SPI) or soy proteinconcentrate (SPC) using methods known in the art. The remaining proteincan be used for producing soy resin (Kim, J. T.; Netravali, A. N.(2010). Effect of protein content in soy protein resins on theirinterfacial shear strength with ramie fibers. Journal of adhesionscience and technology, 24, 203-215).

5.5 Method for Producing BC

Depending on the desired yield, laboratory (small scale) or industrialscale standard methods of bacterial cell culture can be used. Thebacterial strain can be inoculated into the culture medium in a suitablecontainer containing culture medium as the seed culture. The initial pHvalue of the medium can be adjusted to 5.0. The seed culture can then beincubated under standard culture conditions (e.g., for 1-2 days), and aportion of the culture can be inoculated into a new culture medium forproduction of BC. The cultivation can then be carried out using (e.g.,at pH of 5.0 and 30° C.) in a static incubator for 5-14 days.

After incubation, BC pellicles produced on the surface of the culturemedium can be harvested and washed using standard methods. For example,the BC pellicles can be washed successively with water and 1% (w/v),aqueous NaOH at 90° C. for 15 min, and then washed with deionized waterto remove all microbial product contaminants. The purified BC pelliclesthat remain after washing can be dried using standard methods until aconstant weight is obtained.

In certain embodiments, BC yield can increase dramatically during theinitial 3-4 days of culture, then the yield growth can decrease afterseveral more days (e.g., 7 days) of culture. The preferred carbonsources, fructose and glucose, will be nearly used up by this time. BCyield, however, can continue to increase during the period of 7 to 10days of culture, but with a relatively lower rate, mainly becauseAcetobacter xylinum start to consume sucrose and other sugars, which areless preferred for BC production. Also, the decreasing volume of themedium has an effect on lowering the production of BC by the bacteria.

In certain embodiments, BC yield in 100 mL SFE medium can reach 255 mgafter 10 days of culture. BC yield in SFE medium can be as high asyields from using fructose and mannitol media which are generallyregarded in the art as two excellent carbon sources for BC production.

Thus according to the methods of the invention, SFE can be used as anexcellent and inexpensive carbon source for BC production. The yield ofBC production with SFE is high and close to or even much better thanthose obtained with other conventional carbon sources, includingmannitol, fructose, glucose, sucrose and raffinose. The economic cost ofthe carbon source is relatively low because SFE is a by-product of soyflour obtained from soybean which is produced in abundance throughoutthe world.

5.6 Production of BC Films Based ‘Green’ Composites Using Resins

Many types of ‘green’ (e.g., non-toxic, sustainable, non-petroleumbased, and/or biodegradable) composites can be produced using themethods disclosed herein. For example, in one embodiment, BC-soy resin(soy protein isolate (SPI)) composite, crosslinked (e.g., glutaraldehyde(GA)-treated) BC-soy resin (SPI) composite, BC-microfibrillated MFC (ornanofibrillated NFC) cellulose composite and BC-MFC (or NFC)-soy resin(SPI) are produced.

In certain embodiments, these ‘green’ composites can take the form ofpellicles (discs, thin sheets or small balls) or pellets (wet condition)and films or pellets (dry condition).

BC films greatly increase the mechanical properties of soy resin (SPI)materials, including modulus (stiffness) and tensile strength. MFC canbe used to further enhance this effect.

In one embodiment, BC-MFC-soy resin (SPI) composites with higher moduliare provided.

In another embodiment, GA can be used for crosslinking SPI, usingstandard methods, to produce BC-soy resin (SPI) composites withextremely high moduli and better thermal stability than pure BC.

In one embodiment, a method is provided for producing films of BC based‘green’ composites comprising the step of immersing wet BC pelliclesinto soy resin (SPI) aqueous solutions. BC content (i.e., ratio) inBC-soy resin composites can be adjusted by altering the time fortreatment in the aqueous solution.

In one embodiment of the method, an agent can be added, e.g.,microfibrillated cellulose (MFC), nanofibrillated cellulose (NFC),cellulose nanocrystals, cellulose nanowhiskers, nanoparticles, nanoclay,nanocubes (e.g., halloysite), etc. can added into the bacterianutritional medium during fermentation/culture. During BC synthesis, theagents can be interwoven or intercalated into the BC. The BC-agent-soyresin (SPI) composite produced can have different mechanical properties,e.g., higher modulus (stiffness) than composites not comprising theagent.

To prepare a BC-MFC pellicle, MFC (e.g., 5% by weight) can be added intothe culture medium, e.g., a mannitol culture media, to form homogeneousMFC-containing mannitol culture medium.

5.7 Preparation of Soy Resin (SPI) Solutions and Soy Resin (SPI) Sheets

Methods for producing BC-soy resin based composites are provided. In oneembodiment, the method comprises preparing a soy protein isolate (SPI)solution. The SPI solution can be prepared using standard methods, e.g.,by mixing SPI with water (e.g., deionized or tap water) in a ratio of,e.g., 1:15. The ratio can be between 1:10 to 1:25, depending on theviscosity desired.

Glycerol may be added (e.g., 15% by weight) as a plasticizer, and the pHvalue of the solution can be maintained at 8.5 by addition of sodiumhydroxide (Netravali, A. N., Huang, X. and Mizuta, K., Advanced GreenComposites, Advanced Composite Materials, 16, pp. 269-282, 2007; Huang,X.; Netravali, A. N. (2006). Characterization of nano-clay reinforcedphytagel-modified soy protein concentrate resin. Biomacromolecules, 7,2783-2789; Huang, X.; Netravali, A. N. (2007). Characterization of flaxfiber reinforced soy protein resin based green composite modified withnano-clay reinforced. Composites Science and Technology, 67, 2005-2014).The solution can be maintained, e.g., at 95° C. while stirringcontinuously for 40 min, to obtain pre-cured soy resin (SPI) solution.This ‘precuring’ process helps to denature the globular protein byopening up the molecules. Pre-cured soy resin (SPI) can be cast anddried using standard methods, e.g., cast on Teflon® coated glass platesand dried in a 35° C. air circulated dry oven for 16 hr. Dried soy resin(SPI) sheet can be cured using standard methods, e.g., using a CarverHydraulic hot press (model 3981-4PROA00, Wabash, Ind.) at 120° C. for 25min under a pressure of 12.5 MPa. The cured soy resin (SPI) sheet canthen be conditioned at ASTM conditions, e.g., 21° C. and 65% relativehumidity for 3 days. The resins sheets can be assessed for their tensileproperties using standard methods known in the art.

5.8 Preparation of BC-Soy Resin (SPI) and BC-MFC-Soy Resin (SPI)Composites

BC-soy resin (SPI) composites with different BC contents are provided.Such composites can be produced by using BC pellicles that areimpregnated with soy resin (SPI) using ultrasonication for varyingperiods of time, e.g., for 2 hr, 3 hr, 8 hr and 12 hr. The wet BC-soyresin (SPI) composites can be dried using standard methods, e.g., in a35° C. air circulating oven for 8 hr to obtain prepregs. The BC contentin the BC-soy resin (SPI) will be, in certain embodiments, around 20%,25%, 40%, and 50%, according to 2 hr, 3 hr, 8 hr and 12 hr treatmentsrespectively.

To further modify the tensile properties of BC-soy resin (SPI), MFC canbe added into the BC pellicle during the culture to produce BC-MFC-soyresin (SPI). Addition of MFC can increase tensile strain properties ofBC. To produce BC-MFC-soy resin (SPI) composite, BC-MFC pellicle canimpregnated into soy resin (SPI) using standard ultrasonication methods(e.g., for 12 hr). The wet BC-MFC-soy resin (SPI) composite can be driedusing standard methods (e.g., in a 35° C. air circulating oven for 8 hr)to obtain pre-impregnated composite fibers (“prepregs”). The BC-MFCcontent in the BC-MFC-soy resin (SPI) will be, in certain embodiments,approximately 50%.

Prepregs can be cured using standard hot pressing methods (e.g., at 120°C. under a pressure of 12.5 MPa). The cured composites can beconditioned at ASTM conditions (e.g., 21° C. and 65% relative humidityfor 3 days) prior to characterizing their tensile properties.

5.9 Crosslinked BC-Soy Resin (SPI) Composite

Crosslinking with a crosslinking agent can be performed on a BC-resincomposite to make the composite stiffer, more insoluble, stronger, or todecrease the fracture strain or moisture absorption of the composite.Many suitable crosslinking agents are known in the art. Glutaraldehyde(GA) or glyoxal can be applied, using methods known in the art, as acrosslinking agent to a BC-resin composite, to raise the modulus of thecomposite. In other embodiments, rutin or quercetin can be used tocrosslink amine groups in proteins in the BC-resin composite (Huang, X.;Netravali, A. N. (2006). Characterization of nano-clay reinforcedphytagel-modified soy protein concentrate resin. Biomacromolecules, 7,2783-2789; Huang, X.; Netravali, A. N. (2007). Characterization of flaxfiber reinforced soy protein resin based green composite modified withnano-clay reinforced. Composites Science and Technology, 67, 2005-2014).In other embodiments, crosslinkers that react with carboxyl groups,e.g., hydroxyls, diols, ethylene glycol, can be used to crosslinkBC-resin composites.

For example, wet BC-soy resin (SPI) pellicles (BC and PVA ratio=1:1) canbe immersed into GA aqueous solution with a concentration of e.g., 2.5%,5%, 7.5% or 10% (v/v). After 2 hr treatment, GA treated BC-soy resin(SPI) pellicles can be washed with water to remove the residual GA, anddried using standard methods (e.g., in a 35° C. air circulating oven for8 hr) to obtain prepregs. The prepregs can then be cured by standardmethods of hot pressing (e.g., at 120° C. under a pressure of 12.5 MPa).The cured composites were conditioned at ASTM conditions (e.g., 21° C.and 65% relative humidity for 3 days) prior to characterizing theirtensile properties.

The modulus values in GA treated BC-soy resin (SPI) are raiseddramatically (e.g., from 1293.76 MPa to around 3600 MPa) and the valuesof tensile strength and tensile strain are lowered (e.g., from 51.02 MPaand 6.22% to around 46 MPa and 1.40%) compared to those of BC-soy resin(SPI) samples without GA treatment.

5.10 Production of BC-Modified Fibers

Methods for producing fibers surface modified (e.g., coated) withbacterial cellulose (BC) are also provided. BC can be used to modifynatural fibers surfaces and improve the interfacial adhesion betweenfiber and polymer resins, thereby forming truly ‘green’ fiber-reinforcedcomposites with enhanced properties and much better durability.

Any fiber known in the art can be modified with BC. The fiber cancomprise a natural cellulose-based or protein-based material selectedfrom the group of cellulose-based materials consisting of cotton, linen,sisal, ramie, hemp, and bamboo, or from the group of protein-basedmaterials consisting of wool, silk, angora, cashmere, mohair, alpaca,milk protein or soy protein.

The fiber can comprise a polymeric material such as cellulose acetate,nylon, rayon, modacrylic, olefin, acrylic, polyester, polylactic acid,polylactic-co-glycolic acid (PLGA), polyurethane, aramid (e.g. KEVLAR®),ultrahigh molecular weight polyethylene, (e.g. SPECTRA® or DYNEEMA®).The fiber can comprise carbon (e.g., carbon fiber) or glass (e.g.,fiberglass).

Interfacial adhesion between BC-modified fibers and resins (e.g., SPI orsoy resin) can be higher than that between corresponding untreatedfibers and resins. The higher interfacial shear strength (IFSS) likelyarises from the increase in roughness associated with the presence ofnanoscale BC on the fiber surface and the potential for hydrogen bondingbetween the hydroxyl group present on the BC-modified fiber surface andfunctional groups in the resin.

Fiber surfaces may be modified so that BC surrounds the fibers, toimprove their adhesion (interfacial properties) to a variety of resins,thereby improving the composite properties.

In certain embodiments, composites with high IFSS are provided, whichare stronger and stiffer. In other embodiments, composites with low IFSSare provided, which are weaker but tougher.

In one embodiment, the method for producing BC-modified fibers cancomprise inoculating a bacterial strain into a mannitol culture medium(or other suitable sugar culture medium as described herein) as the seedculture. The seed culture is incubated using standard culture methods asdescribed herein, and inoculated into a fiber-sugar bacteria nutritional(culture) medium for production of BC-fiber composite. The cultivationis carried out using standard culture methods. After cultivation, thesurface of the fiber will be coated a layer of BC. The BC-modified fibercan then be harvested and washed successively with water and 1% (w/v),aqueous NaOH at 90° C. for 15 min, and then washed by deionized water toremove all microbial product contaminants.

5.11 Preparation of Resin

Any resin known in the art can be used in the methods and compositionsdisclosed herein. In a specific embodiment, SPI or soy resin can bemixed with deionized or tap water in a ratio of, e.g., 1:10 to 1:20. Themixture can be homogenized and then ‘pre-cured’ (e.g., maintaining themixture in a water bath at 75° C. for an 30 min). This precuring processhelps denature the globular protein by opening up the molecules. Thepre-cured resin can be used to make microbeads on a single fiber.

In other embodiments, BC-modified fibers can be impregnated intopre-cured resin and then clamped at both ends to ensure a high degree ofalignment during composite fabrication. FIG. 12 shows a BC-Sisal-soyresin hybrid composite.

To obtain BC-modified fiber-resin with greater alignment of the fibers,silicon molds made by standard methods can be used, as shown in FIG. 13.

IFSS of the BC-modified fiber can be tested using the art-knownmicrobond test (FIG. 9). The fiber diameter, d and embedded length, L,can be measured prior to the microbond test using a calibrated opticalmicroscope. To obtain accurate measurements, d and L are measured againafter the microbond test. The microbond test can be performed usingstandard, commercially available instrumentation (e.g., an Instronuniversal testing machine, model 5566 with a microvise). The microviseplates are placed just above a microbead and brought closer until theybarely touched the fiber surface as shown in FIG. 9. The fiber beingtested is then pulled out from the microbead until the microbeaddebonds. The interfacial shear strength, τ, is calculated using thefollowing equation:

${{IFSS}(\tau)} = \frac{F}{\pi \times d \times L}$

where F is the force required to debond the microbead. For purposes ofcalculation, it can be assumed that the shear strength is uniform alongthe entire fiber/microbead interface.

5.12 Production of Films BC-Based Biocompatible Polymer Composites

Water-soluble and biocompatible (and/or biodegradable) polymers can beemployed in the methods for producing ‘green’ composites. They enhancethe properties of BC and form soft, uniform, and biocompatiblecomposites. Poly (vinyl alcohol) (PVA) and poly (ethylene oxide) (PEO)are two kinds of thermoplastic polymers that are water-soluble,nonvolatile and biocompatible. PVA is also biodegradable. The hydrogenbonds are formed between hydroxyl groups of BC and PVA or PEO, thusfurther improving the uniformity and strength of the composites.

In specific embodiments, the BC-based water-soluble and biocompatiblepolymer composites produced according to the methods of the inventionare BC-PVA and BC-PEO composites.

BC content in BC-resin composites can be adjusted by using differentconcentrations of aqueous solutions of the resin. The BC content canvary between 20 to 60% (by weight) of the BC-resin composite, dependingon the desired application, with higher BC content resulting in strongercomposites.

The inclusion of BC in resin materials can greatly increase theirmechanical properties, including modulus (stiffness) and tensilestrength, because hydrogen bonds can form between hydroxyl groups of BCand the resin. The BC-based water-soluble and biocompatible polymercomposites such as BC-PVA and BC-PEO have better thermal stability thanthat of pure BC. These composites have smooth surfaces and uniformthicknesses. Resin not only penetrates into the BC network, but alsofills in pores among the nanofibers formed by the BC network.

In other embodiments, BC can be used to make membrane-like compositesthat can be used “as is” or can be added to another composite to improvethe properties of the composite. In addition to PVA and PEO, other‘green’ or biodegradable resins such as soy protein resins or starch canbe used.

In other embodiments, ‘non-green’ resins such as epoxies, unsaturatedpolyester, polyurethane, vinyl ester, etc. can also be used. In certainembodiments, a resin can be transparent, and the resulting BC-modifiedresin can be used in applications in which strong or durable transparentmaterials such as glass or transparent thermoplastic are generally used.

In one embodiment, a method is provided for producing films of BC-based‘green’ composites comprising the step of immersing wet BC pelliclesinto an aqueous resin solution. The BC-based composites produced by themethod have smoother surfaces and more uniform thicknesses, and theirmechanical properties and thermal properties are better than compositespreviously achieved in the art.

5.13 Methods for Producing Films of BC-Resin Composites

Methods for producing films of BC-resin composites are provided. In oneembodiment, an aqueous solution of resin powder (e.g., PVA powder) isprepared using standard methods. Purified BC pellicles are immersed intothe resin solution under standard conditions known in the art (e.g., inan 80° C. water bath for 2 hr). The BC pellicles are immersed in theresin solution at room temperature (e.g., for 12 hr). Theresin-containing BC pellicle is then transferred into deionized waterfor 30 min to remove superfluous resin on the surface of the BC pellicleand to stabilize the BC-resin composite. The film of BC-resin compositeis dried using standard methods until a constant weight was obtained.

In one embodiment, the mean diameter of BC-modified nanofibers is lessthan 100 nm and the diameter of pore ranges from several dozens toseveral hundred nanometers.

5.14 Methods for Characterizing Bacterial Cellulose (BC) Based ‘Green’Composites

BC based green composites can be characterized using standard methods ofscanning electron microscopy (SEM), tensile testing, moisture contenttesting, etc.

Samples of BC based green composites can be characterized by a Fouriertransform infrared spectrometer for the evaluation of chemicalstructures.

Thermogravimetric analysis (TGA, TA instrument) can carried out tocharacterize the thermal properties of samples.

Interfacial shear strength (IFSS) between BC-modified fibers and resinscan be tested by using the art-known methods, such as the microbondtest.

5.15 Uses for Bacterial Cellulose Based ‘Green’ Composites

The BC based green composites can have many uses, including but notlimited to: components of electronics (circuit boards), components ofmicrophones and speakers (e.g., diaphragms), wound dressings, scaffoldsfor tissue engineering, synthetic dura mater (brain covering), bladderneck suspension, soft tissue replacement, artificial blood vessels, dietfoods and other foods (e.g., nata de coco), matrices for electronicpaper, reinforcement for paper, tape or other adhesives, automotivecomponents (e.g., automobile body, many reinforced plastic body parts),aerospace components (airplanes, rockets, etc.), building materials(construction poles, walls, etc.) to sporting goods (tennis or badmintonrackets, ski poles, fishing rods, etc.), packaging and otherapplications in which biodegradable membranes are desirable.

In other embodiment, fully transparent composites (made with transparentresins) can be used in place of glass in many applications to reduceweight (e.g., buildings, automobiles, airplanes, etc.).

The following examples are offered by way of illustration and not by wayof limitation.

6. EXAMPLES 6.1 Example 1 Low Cost Carbon Source from Soy Flour forBacterial Cellulose (BC) Production by the Acetobacter xylinum

This example demonstrates the successful development from defatted soyflour of a low-cost carbon source for production of bacterial cellulose(BC) using Acetobacter xylinum. Soy flour extract consists of severalsugars such as fructose, glucose, sucrose, raffinose and stachyose. Allof them can be used as carbon source for bacterial cellulose production.The example demonstrates that Acetobacter xylinum prefer consumingsugars in the following order: fructose and glucose, sucrose, andraffinose and stachyose during the culture process. Results alsoindicated that the autoclaving process resulted in splitting sucrose tofructose and glucose. Based on the same concentration of sugars (25 g/L)in the culture media, bacterial cellulose yield using soy flour extractmedium (based only on the concentration of fructose, glucose andsucrose) was close to or even higher than the yields obtained usingconventional carbon sources media such as glucose, fructose andmannitol.

Introduction

A low-cost carbon source, soy flour extract (SFE), was developed fromsoy flour (SF) for BC production. The sugars were extracted bysolubilizing them in water while keeping the protein insoluble (Kim, J.T.; Netravali, A. N. 2010. Mechanical, Thermal, and InterfacialProperties of Green Composites with Ramie Fiber and Soy Resins. Journalof Agricultural and Food Chemistry. DOI: 10.1021/jf100317y.)

The method of SFE preparation was extremely inexpensive and convenientand the results indicated that the BC production yield was comparable tothose obtained by other researchers using other carbon sources. Theconsumption of different sugars by Acetobacter xylinum and thecompositional changes of sugars in the SFE medium during autoclavingwere analyzed as well.

Materials and Methods

Microorganism and Culture Media

Acetobacter xylinum, ATCC 23769, obtained from the American Type CultureCollection (ATCC, Manassas, Va.) was used as the model strain andmaintained on agar plates containing 25 g/L D-mannitol, 5 g/L yeastextract and 5 g/L tryptone and 20 g/L agar. The SFE medium used for BCproduction consisted of 5 g/L yeast extract, 5 g/L tryptone and theautoclaved SFE as the sole carbon source. Other culture media used forcomparison of BC yields consisted of 25 g/L carbon sources (includingraffinose, glucose, sucrose, fructose and mannitol respectively), 5 g/Lyeast extract and 5 g/L tryptone.

Treatment of Soy Flour Powder

Soy flour (7B) powder obtained from ADM Co. (Decatur, Ill.) was mixedwith deionized water to produce soy flour extract (SFE). Soy flourpowder was initially soaked in deionized water in a ratio of 3:17 and pHvalue of the mixture was adjusted to 4.5 by adding hydrochloric acid.The mixture was maintained at 50° C. in a water bath for 1 hr. Afterthat, the mixture was filtered to remove the solid contents, mostly theinsoluble protein. Part of the filtrate was then allowed to evaporate toobtain the desired suitable main sugar concentration (based only on theconcentration of fructose, glucose and sucrose) which was around 25 g/L(preferred concentration) for BC culture. Other main sugarconcentrations between 15 and 30 g/L can also be used (with lowerconcentration resulting in lower BC production and vice versa).

Consumption of Sugars in SFE Medium

The concentrations of sugars, including fructose, glucose, sucrose,raffinose and stachyose were determined before and after autoclavingusing art-known methods of high performance liquid chromatography (HPLC)using an UltiMate 3000 LC system (Dionex, Sunnyvale, Calif.) attachedwith the refractive index (RI) detector (RI-101, Ecom, Purage, CzechRepublic). Autoclaving of the SFE was carried out at 121° C. and 15 psiin a sterilizer (Market forge, Alfa Medical, Westbury, N.Y.) for 25 min.After removing big protein deposits, the autoclaved SFE was used for BCculture. Sugar concentrations in the SFE during 10 days of culture weredetermined on a daily basis using HPLC. After filtering the samplesthrough a 0.45 μm pore size polytetrafluoroethylene (PTFE) filter andremoving tiny BC fibrils and other impurities in the SFE medium, eachsugar concentration was analyzed with a SUPELCOSIL LC-NH₂ column (25cm×4.6 mm ID and 5 μm particles, Supelco, Bellefonte, Pa.) and the RIdetector. The HPLC column was used at 30° C. temperature. The mobilephase was the mixture of acetonitrile and deionized water (3:1, v/v) andwas maintained at a flow rate of 1 ml/min.

Production of BC

The strain was inoculated into a conical flask containing the abovementioned SFE culture medium as the seed culture. The initial pH valueof the medium was adjusted to 5.0 and was not regulated during theculture. The seed culture was incubated at 30° C. and 130 rpm on arotary shaker for 2 days, and 6 mL of this was inoculated into a 100-mLculture medium in a 600 ml conical flask for production of BC. Thecultivation was carried out initially at pH of 5.0 and 30° C. in astatic incubator for 10 days. Samples were taken from the SFE mediumevery day during the 10-day culture to measure the consumption of sugarsand BC yields.

BC Harvesting and Weighing

After incubation, the BC pellicles produced on the surface of SFE mediumand other culture media mentioned in section 2.1 were harvested everyday and washed successively with water and 1% (w/v), aqueous NaOH at 90°C. for 15 min, and then washed by deionized water to remove allmicrobial product contaminants. The remaining purified cellulosepellicles were finally dried at 105° C. on a Teflon® plate untilconstant weight was obtained. BC pellicles cultured in SFE medium andother culture media mentioned in section 2.1 were compared for theiryields.

Results and Discussion

Influence of Autoclaving on SFE Medium

The HPLC analysis of the crude SFE used in this study showed that itconsisted of 1.92 g/L fructose and glucose (combined), 21.21 g/Lsucrose, 1.59 g/L raffinose, 11.92 g/L stachyose, water and othercomponents including proteins. The concentration of total sugars wasover 36 g/L. After autoclaving (sterilizing) at 121° C. and 15 psi for25 min, however, the HPLC analysis showed a slightly differentcomposition of sugars in the SFE medium. Therefore, the influence ofautoclaving on the SFE medium was investigated.

Sucrose is prone to heat degradation during autoclaving and that thesucrose-containing media sterilization will result in a mixture ofD-glucose, D-fructose and sucrose (Dobbs, J. H.; Roberts, L. W. (1995).Experiments in plant tissue culture, third edition. Cambridge Press, pp.53). 15% to 25% of the sucrose may hydrolyze to glucose and fructoseduring autoclaving at the elevated temperature (Ball, E. (1953).Hydrolysis of Sucrose by Autoclaving Media, a Neglected Aspect in theTechnique of Culture of Plant Tissues. Bulletin of the Torrey BotanicalClub, 80(5), 409-411; Schenk, N.; Hsiao, K. C.; Bornman, C. H. (1991).Avoidance of precipitation and carbohydrate breakdown in autoclavedplant tissue culture media. Plant Cell Reports, 10(3), 115-119).

Table 1 presents the HPLC data for various sugar concentrations in SFEbefore and after autoclaving. Table 1 also gives adjusted values for allsugars after considering the water evaporation during autoclaving.Before autoclaving, the crude SFE medium had concentrations of 1.92 g/Lfor fructose and glucose, 21.21 g/L for sucrose, 1.59 g/L for raffinose,and 11.92 g/L for stachyose. After the autoclaving the concentrationschanged to 7.54 g/L for fructose and glucose, 17.54 g/L for sucrose,1.58 g/L for raffinose and 9.92 g/L for stachyose. The concentration oftotal sugars was 36.58 g/L and the concentration of three traditionalcarbon sources (fructose, glucose and sucrose) for BC production wasapproximately 23-25 g/L which was almost the same as the regularconcentration of carbon source used for BC production by others (Hong,F.; Qiu, K. (2008). An alternative carbon source from konjac powder forenhancing production of bacterial cellulose in static cultures by amodel strain Acetobacter aceti subsp. xylinus ATCC 23770. CarbohydratePolymers, 72 (3), 545-549).

TABLE 1 Effect of autoclaving on the concentrations of various sugars inthe SFE medium Concentrations of sugars in SFE medium Fructose and TotalGlucose Sucrose Raffinose Stachyose Sugars (g/L) (g/L) (g/L) (g/L) (g/L)SFE before 1.92 21.21 1.59 11.92 36.64 autoclave SFE after 7.54 17.541.58 9.92 36.58 autoclave SFE after 7.16 16.66 1.50 9.42 34.74 autoclave(adjusted data by considering water evaporation factor)

The data in Table 1 indicate that there was a significant decrease insucrose concentration and an almost equivalent increase in the fructoseand glucose concentration after autoclaving. As a result, there was nochange in the combined concentration of fructose, glucose and sucrosewhich remained in the range of 23-25 g/L, before and after autoclaving.As discussed earlier, this was mainly due to the degradation of sucrose(Dobbs, J. H.; Roberts, L. W. (1995). Experiments in plant tissueculture, third edition. Cambridge Press, pp. 53; Ball, E. (1953).Hydrolysis of Sucrose by Autoclaving Media, a Neglected Aspect in theTechnique of Culture of Plant Tissues. Bulletin of the Torrey BotanicalClub, 80(5), 409-411; Schenk, N.; Hsiao, K. C.; Bornman, C. H. (1991).Avoidance of precipitation and carbohydrate breakdown in autoclavedplant tissue culture media. Plant Cell Reports, 10(3), 115-119). Thedata also indicated that the concentration of stachyose and raffinosedecreased a little during the autoclaving process (Table 1). This may beowing to hydrolysis of raffinose and stachyose similar to that ofsucrose. In experiments on pure rafffinose and stachyose, they were notobserved to degrade during the short time of autoclaving (althoughhydrolysis is a possibility). During autoclaving, raffinose andstachyose may hydrolyze as sucrose does. The reduction of raffinose andstachyose in SFE medium during autoclaving may be also due to a sidereaction such as caramelization or maillard reaction. Other suitabletemperature and pressure conditions for autoclaving can be easilydetermined by one of skill in the art. In certain embodiments, highertemperatures will produce faster sugar degradation.

Sugar Consumption in SFE Medium During Culture

All five sugars in SFE, fructose, glucose, sucrose, raffinose andstachyose, could be used as carbon sources separately for BC culture byAcetobacter xylinum and different sugars had different effectiveness forBC yields. To measure the actual consumption of individual sugars in theculture medium was analyzed for the sugar content every day.

FIG. 1 shows plots of the change in concentrations of all five sugars asa function of culture time in days. Based on the plots, fructose andglucose concentration decreased steadily and almost linearly until day7. During that period the other sugar concentrations remained more orless stable. After the sixth day the sucrose concentration started todecrease. When fructose and glucose concentration decreased to arelatively low value (around 1.09 g/L) from initial 7.54 g/L, theAcetobacter xylinum started to consume sucrose. These results indicatethat the Acetobacter xylinum preferred to consume fructose and glucosebefore the other three sugars present in the SFE medium. During theentire 10-day culture time, very little or no raffinose and stachyosewere consumed and no significant change was noticed in theirconcentrations.

BC Yield in SFE Medium

BC yield in fructose or glucose media is higher than in sucrose medium(Yang, Y. K.; Park, S. H.; Hwang, J. W.; Pyun, Y. R.; Kim, Y. S. (1998).Cellulose production by Acetobacter xylinum BRCS under agitatedconditions. Journal of Fermentation and Bioengineering, 85(3), 312-317).The plot of BC yield in the data presented FIG. 2 confirms that fructoseand glucose were better carbon sources for BC production as comparedwith sucrose, raffinose and stachyose, if the pH value of the media werekept constant. After autoclaving, the fructose and glucose concentrationin the SFE medium reached 7.54 g/L from the initial concentration of1.92 g/L. The higher fructose and glucose concentration was beneficialfor BC culture. It is important to note that the concentration of threesugars (fructose, glucose and sucrose) in the SFE medium, thatAcetobacter xylinum mainly consumed during the 10-day culture, wasaround 25 g/L which was the same as the regular concentration ofconventional carbon sources used by other researchers for BC production(Hong, F.; Qiu, K. (2008). An alternative carbon source from konjacpowder for enhancing production of bacterial cellulose in staticcultures by a model strain Acetobacter aceti subsp. xylinus ATCC 23770.Carbohydrate Polymers, 72 (3), 545-549).

FIG. 2 shows a plot of BC yield in SFE medium as a function of culturetime in days. As seen in FIG. 2, BC yield increased dramatically duringthe initial 3-4 days and then the yield growth significantly decreasedafter 7 days' of culture. The main reason for this was that thepreferred carbon sources, including fructose and glucose, were almostused up at this time. BC yield, however, continued to increase duringthe period of 7 to 10 days but with a relatively lower rate becauseAcetobacter xylinum started to consume sucrose and other sugars whichwere not as suitable as fructose and glucose for BC production. Theresults in FIG. 2 also indicate that BC yield in SFE can reach 255 mgafter 10 days of culture and this value is close to or even better thanBC yields with other conventional carbon sources under similar cultureconditions (Keshk, S.; Sameshima, K. (2005). Evaluation of differentcarbon sources for bacterial cellulose production. African Journal ofBiotechnology, 4(6), 478-482; Hong, F.; Qiu, K. (2008). An alternativecarbon source from konjac powder for enhancing production of bacterialcellulose in static cultures by a model strain Acetobacter aceti subsp.xylinus ATCC 23770. Carbohydrate Polymers, 72 (3), 545-549). FIG. 3compares BC yields by using different carbon sources individually after10 days of culture. BC yield in SFE medium (255 mg) was almost as highas those using fructose (270.3 mg) and mannitol (276.3 mg) media whichpreviously were regarded as two excellent carbon sources for BCproduction. Based on the data, it was concluded that SFE could be usedas an excellent and one of the least expensive carbon sources for BCproduction. BC yield in SFE medium was also much higher than those inraffinose (29.7 mg), sucrose (72.8 mg) and glucose (128.2 mg) media.Glucose was reported as an excellent carbon source for BC production(Keshk, S.; Sameshima, K. (2005). Evaluation of different carbon sourcesfor bacterial cellulose production. African Journal of Biotechnology,4(6), 478-482) but the yield was lower in our pure glucose mediumbecause the pH value of the medium was not regulated and gluconic acidgenerated by glucose during the culture caused pH value changed to less3.5 which was not suitable for BC production. However, the glucose inSFE medium still could be used as a good carbon source because pH valuein SFE medium did not change significantly during the culture. Thereason might be that relatively low amount of gluconic acid was formedpossibly due to several other sugars being present creating a buffereffect in the SFE medium.

The BC yield in SFE medium (255 mg) was also higher than previouslyreported BC yields by using konjac powder hydrolyzate (212 mg,Acetobacter xylinum-ATCC 23770, 8 days) and processed rice bark (242 mg,Acetobacter xylinum-ATCC 23769, 10 days) (Hong, F.; Qiu, K. (2008). Analternative carbon source from konjac powder for enhancing production ofbacterial cellulose in static cultures by a model strain Acetobacteraceti subsp. xylinus ATCC 23770. Carbohydrate Polymers, 72 (3), 545-549;Goelzer, F. D. E.; Faria-Tischer, P. C. S.; Vitorino, J. C.;Sierakowski, Maria-R.; Tischer, C. A. Production and characterization ofnanospheres of bacterial cellulose from Acetobacter xylinum fromprocessed rice bark. (2009). Materials Science and Engineering, C:Materials for Biological Applications, 29(2), 546-551).

Conclusion

The results of this example demonstrate that SFE can be an excellentcarbon source for BC production. The yield of BC production with SFE washigh and close to or even much better than those obtained with otherconventional carbon sources, including mannitol, fructose, glucose,sucrose and raffinose. The economic cost of the carbon source isrelatively low because SFE is a by-product of soy flour obtained fromsoybean which is produced in abundance throughout the world.

The example also demonstrates that SFE contains at least five sugars andall of them can be used as carbon sources for BC production. Inaddition, the example demonstrates that autoclaving process can resultin the change of the composition of sugars in SFE owing to hydrolysis ofhigher sugars.

The results also demonstrate that Acetobacter xylinum prefers to consumefructose and glucose before sucrose and other carbon sources during theculture. When the concentration of fructose and glucose is relativelylow, Acetobacter xylinum then starts to consume sucrose more. Althoughit was seen that Acetobacter xylinum can consume raffinose andstachyose, the consumption rates were very low. Moreover, the resultsshow that the rate of BC production is much higher when theconcentration of fructose and glucose is high.

6.2 Example 2 Production of BC Films Based ‘Green’ Composites by UsingSoy Resin

This example demonstrates the production of films of bacterial cellulose(BC) based ‘green’ composites, including BC-soy resin (soy proteinisolate (SPI)) composite, glutaraldehyde (GA)-treated soy resin (SPI)composite, BC-microfibrillated cellulose (MFC) composite and BC-MFC-soyresin (SPI). These composites had smooth surfaces and uniformthicknesses. SEM images indicated that MFC penetrated into the networkof BC nanofibers. Tensile test indicated that BC films could greatlyincrease the mechanical properties of soy resin (SPI) materials,including modulus and tensile strength. MFC could further enhance thiseffect, so that BC-MFC-soy resin (SPI) composites with higher moduliwere obtained. Also, an extremely high modulus for BC-soy resin (SPI)composite could be achieved by using GA for crosslinking FTIR testingshowed the presence of amino and carboxyl groups in BC-soy resin (SPI)and BC-MFC-soy resin (SPI) composites. TGA showed that BC-soy resin(SPI) composites had better thermal stabilities than pure BC.

Introduction

During past several decades, new advanced composites with excellentmechanical properties have been developed and used as metalreplacements. However, most composites are made using syntheticnon-degradable fibers, such as carbon, aramid and glass and polymers(resins), such as polyetheretherketone (PEEK) and epoxy. Thesecomposites pose serious solid waste disposal problems due to decreasinglandfill space, widespread litter and pollution of marine environments(Chou, T. W., Frank, K. K. (1989) Composite materials series, 3, Textilestructural composites. Elsevier science publishers, New York, 1-26;Mohanty, A. K.; Khan, Mubarak A.; Hinrichsen, G. (2000) Surfacemodification of jute and its influence on performance of biodegradablejute—fabric/Biopol composites. Composites science and technology, 60(7), 1115-1124). As a result, biodegradable and bio-based materials haveattracted much attention in recent years. ‘Green’ composites have beenpreviously fabricated using soy based resins reinforced with liquidcrystalline high strength cellulose fibers (Netravali, A. N., Huang, X.and Mizuta, K., Advanced Green Composites, Advanced Composite Materials,16, pp. 269-282, 2007).

Bacterial cellulose (BC) is a promising biodegradable material withbroad potential for composite reinforcement. BC is can be produced byAcetobacter xylinum, a Gram-negative, obligately aerobic bacterium, in anutritional fermentation medium at 30° C. for several of days. BC hasthe same chemical structure as other plant-based cellulose. However, BCis composed of fiber diameters of only few nanometers and displays manyunique properties, including higher purity, higher crystallinity, higherdegree of polymerization, higher tensile strength, higher modulus andstronger biological adaptability (Iguchi, M.; Yamanaka, S.; Budhiono, A.(2000) Bacterial cellulose—a masterpiece of Nature's arts. Journal ofmaterials science, 35 (2), 261-270; Baeckdahl, H.; Helenius, G.; Bodin,A.; Nannmark, U.; Johansson, B. R.; Risberg, B.; Gatenholm, P. (2006).Mechanical properties of bacterial cellulose and interactions withsmooth muscle cells. Biomaterials, 27 (9), 2141-2149; Klemm, D.;Schumann, D.; Udhardt, U.; Marsch, S. (2001). Bacterial synthesizedcellulose—artificial blood vessels for microsurgery. Progress in polymerscience, 26 (9), 1561-1603; Klemm, D.; Heublein, B.; Fink, H. P.; Bohn,Andreas. (2005). Cellulose: Fascinating biopolymer and sustainable rawmaterial. Angewandte chemie, International edition, 44(22), 3358-3393;Fink, H. P.; Weigel, P.; Purz, H. J.; Ganster, J. (2001). Structureformation of regenerated cellulose materials from NMMO-solutions.Progress in Polymer Science, 26(9), 1473-1524).

BC materials have been used in a variety of applications, includingartificial skin and blood vessels, binding agents, loud speakerdiaphragms, paper, foods, textiles, composite membranes, etc. (Wan, Y.;Hong, L.; Jia, S.; Huang, Y.; Zhu, Y.; Wang, Y.; Jiang, H. (2006).Synthesis and characterization of hydroxyapatite—bacterial cellulosenanocomposites. Composites science and technology, 66 (11-12),1825-1832; Fontana, J. D.; De Souza, A. M.; Fontana, C. K.; Torriani, I.L.; Moreschi, J. C.; Gallotti, B. J.; De Souza, S. J.; Narcisco, G. P.;Bichara, J. A.; Farah, L. F. X. (1990). Acetobacter cellulose pellicleas a temporary skin substitute. Applied Biochemistry and Biotechnology,24-25, 253-264; Shibazaki, H.; Kuga, S.; Onabe, F.; Usuda, M. (1993).Bacterial cellulose membrane as separation medium. Journal of appliedpolymer science, 50 (6), 965-9; Svensson, A.; Nicklasson, E.; Harrah,T.; Panilaitis, B.; Kaplan, D. L.; Brittberg, M.; Gatenholm, P. (2005).Bacterial cellulose as a potential scaffold for tissue engineering ofcartilage. Biomaterials, 26 (4), 419-431). Dry BC material is not suitedfor some applications, however, owing to its mechanical and thermalproperties.

Soy proteins are biodegradable polymers and have been used in ‘green’composites because of their worldwide availability and low price. Amongsoy proteins, soy protein isolate (SPI) is a highly refined or purifiedform with minimum protein content of 90% on a moisture-free basis. It ismade from defatted soy flour which has had most of the non-proteincomponents, fats and carbohydrates removed. SPI contains 18 differentamino acids which have polar functional groups such as hydroxyl, amineand carboxyl groups. These functional groups have the potential to reactwith, or hydrogen bond to, natural cellulose fibers that containhydroxyl groups (Kim, J. T.; Netravali, A. N. (2010). Effect of proteincontent in soy protein resins on their interfacial shear strength withramie fibers. Journal of adhesion science and technology, 24, 203-215).

BC-resin composites have been investigated previously (Nakagaito, A. N.;Iwamoto, S.; Yano, H., Bacterial cellulose: the ultimate nano-scalarcellulose morphology for the production of high-strength composites.Applied Physics A: Materials Science and Processing (2004), Volume Date2005, 80 (1), 93-97; Ifuku, S.; Nogi, M.; Abe, K.; Handa, K.; Nakatsubo,F.; Yano, H., Surface Modification of Bacterial Cellulose Nanofibers forProperty Enhancement of Optically Transparent Composites: Dependence onAcetyl-Group DS. Biomacromolecules (2007), 8 (6), 1973-1978).High-strength composites using BC sheet impregnated with phenolic resinor acrylic resin have been developed (Nakagaito, A. N.; Iwamoto, S.;Yano, H., Bacterial cellulose: the ultimate nano-scalar cellulosemorphology for the production of high-strength composites. AppliedPhysics A: Materials Science and Processing (2004), Volume Date 2005, 80(1), 93-97; Ifuku, S.; Nogi, M.; Abe, K.; Handa, K.; Nakatsubo, F.;Yano, H., Surface Modification of Bacterial Cellulose Nanofibers forProperty Enhancement of Optically Transparent Composites Dependence onAcetyl-Group DS. Biomacromolecules (2007), 8 (6), 1973-1978). Althoughuseful, the resins used in art-known BC-resin composites are notbiodegradable and could cause health or environmental problems.

The inexpensive ‘green’ resins disclosed in this example, such asmodified SP, including SF, soy protein concentrate (SPC), soy proteinisolate (SPI), etc., can be used to reinforce the BC and fabricate‘green’ composites with high mechanical and physical properties in theproposed research.

In this example, films of BC based ‘green’ composites were successfullydeveloped by immersing wet BC pellicles into soy resin (SPI) aqueoussolutions. BC contents in BC-soy resin composites could be adjusted byvarying the treatment time in the aqueous solution.

Microfibrillated cellulose (MFC) was added into BC pellicles duringculture and BC-MFC-soy resin (SPI) composites with higher moduli werealso produced.

In addition, glutaraldehyde (GA) was used as crosslinking agent for soyresin (SPI) to produce GA treated BC-soy resin (SPI) composites withhigher moduli (stiffness).

The BC-green resin composites disclosed in this example had smoothsurfaces and uniform thicknesses. Excellent mechanical properties andthermal properties were achieved.

Materials and Methods

Microorganism and Culture Media

Acetobacter xylinum, ATCC 23769, obtained from the American Type CultureCollection (ATCC, Manassas, Va.) was maintained on agar platescontaining 25 g/L D-mannitol, 5 g/L yeast extract and 5 g/L tryptone and20 g/L agar. The mannitol culture medium used for BC productionconsisted of 25 g/L D-mannitol, 5 g/L yeast extract and 5 g/L tryptone.

Preparation of BC Pellicle

The strain from the agar plate was inoculated into a conical flaskcontaining mannitol culture medium as the seed culture. The initial pHvalue of the medium was adjusted to 5.0 and was not regulated duringsubsequent culture. The seed culture was incubated at 30° C. and 130 rpmon a rotary shaker for 2 days, and 6 mL of seed liquid was inoculatedinto 100-mL of culture medium in 600-mL conical bottle for production ofBC. The cultivation was carried out at an initial pH of 5.0 and at 30°C. in a static incubator for 10 days. After incubation, the BC pellicleproduced on the surface of the mannitol culture medium was harvested andwashed successively with water and 1% (w/v), aqueous NaOH at 90° C. for15 min, and then washed with deionized water to remove all microbialproduct contaminants.

Preparation of BC-MFC Pellicle

MFC (5% by weight) was added into mannitol culture media (describedabove) to form homogeneous MFC containing mannitol culture medium foruse in the production of BC. After 2 days in seed culture (as describedabove), 6 mL of the seed liquid was inoculated into a 100-mLMFC-containing culture medium in 600-mL conical bottle for production ofBC. The cultivation was carried out at an initial pH of 5.0 and at 30°C. in a static incubator for 10 days. After incubation, the BC-MFCpellicle produced on the surface of MFC-containing mannitol culturemedium was harvested and washed successively with water and 1% (w/v),aqueous NaOH at 90° C. for 15 min, and then washed with deionized waterto remove microbial product contaminants. During the entire washingprocess, the BC-MFC pellicle was treated carefully in order to avoidseparation of the MFC and BC pellicle.

Preparation of Soy Resin (SPI) Solution And Soy Resin (SPI) Sheet

Desired soy resin (soy protein isolate (SPI)) was initially mixed withdeionized water in a ratio of 1:15. Glycerol was added (15% by weight)as a plasticizer. The pH value of the solution was maintained at 8.5 byaddition of sodium hydroxide (Netravali, A. N., Huang, X. and Mizuta,K., Advanced Green Composites, Advanced Composite Materials, 16, pp.269-282, 2007; Huang, X.; Netravali, A. N. (2006). Characterization ofnano-clay reinforced phytagel-modified soy protein concentrate resin.Biomacromolecules, 7, 2783-2789; Huang, X.; Netravali, A. N. (2007).Characterization of flax fiber reinforced soy protein resin based greencomposite modified with nano-clay reinforced. Composites Science andTechnology, 67, 2005-2014). The solution was maintained at 95° C. whilestirring continuously for 40 min to obtain pre-cured soy resin (SPI)solution. This ‘precuring’ process helps denature the globular proteinby opening up the molecules. Pre-cured soy resin (SPI) was cast on theTeflon® coated glass plates and dried in a 35° C. air circulated dryoven for 16 hr. Dried soy resin (SPI) sheet was cured using CarverHydraulic hot press (model 3981-4PROA00, Wabash, Ind.) at 120° C. for 25min under a pressure of 12.5 MPa. The cured soy resin (SPI) sheet wasconditioned at ASTM conditions of 21° C. and 65% relative humidity for 3days before tensile testing.

Preparation of BC-Soy Resin (SPI) and BC-MFC-Soy Resin (SPI) Composites

BC-soy resin (SPI) composites with different BC contents were producedby using BC pellicles and impregnating them with soy resin (SPI) usingultrasonication for 2 hr, 3 hr, 8 hr and 12 hr respectively. The wetBC-soy resin (SPI) composites were dried in a 35° C. air circulatingoven for 8 hr to obtain prepregs. The BC content in the BC-soy resin(SPI) were around 20%, 25%, 40% and 50% for impregnations of 2 hr, 3 hr,8 hr and 12 hr respectively.

For BC-MFC-soy resin (SPI) composite production, BC-MFC pellicle wasimpregnated into soy resin (SPI) using ultrasonication for 12 hr. Thewet BC-MFC-soy resin (SPI) composite was dried in a 35° C. aircirculating oven for 8 hr to obtain prepregs (i.e., pre-impregnatedand/or partially cured composite sheets. The BC-MFC content in theBC-MFC-soy resin (SPI) was around 50%.

All the prepregs were then cured by hot pressing at 120° C. under apressure of 12.5 MPa. The cured composites were conditioned at ASTMconditions of 21° C. and 65% relative humidity for 3 days prior tocharacterizing their tensile properties.

Preparation of Glutaraldehyde (GA) Treated BC-Soy Resin (SPI) Composite

Glutaraldehyde (GA) was used as a crosslinking agent for the soy basedresin. Wet BC-soy resin (SPI) pellicles (BC and SPI ratio=1:1) wereimmersed into GA aqueous solutions with concentrations of 2.5%, 5%, 7.5%or 10% (v/v). After 2 hr treatment, the GA treated BC-soy resin (SPI)pellicles were washed with water to remove the residual GA. They werethen dried in a 35° C. air circulating oven for 8 hr to obtain prepregs.The prepregs were then cured by hot pressing at 120° C. under a pressureof 12.5 MPa. The cured composites were conditioned at ASTM conditions of21° C. and 65% relative humidity for 3 days prior to characterizingtheir tensile properties.

Characterization

Scanning electron microscopy (SEM) images of dry BC-MFC film were takenwith a Leica 440 scanning electron microscope. The sample was sputtercoated with gold and morphology was observed by the SEM at anaccelerating voltage of 15 kV.

Tensile testing was performed with a Instron tensile test machine(5566). The test specimens were prepared by cutting the membranes to 10mm wide and 50 mm long strips using a precise cutter. Tensile testingwas conducted according to ASTM D-882-02 as a standard test method fortensile elastic properties of thin plastic sheeting. Two ends of thespecimens were placed between the upper and lower jaws of theinstrument, leaving a gauge length of 30 mm of the specimens between thetwo jaws. Strain rate of the instrument was 2%/min. The Young's modulusof samples was calculated from the tensile test results.

Moisture content (MC) was measured by a commercial moisture/volatiletester (C.W. Brabender Instrument Inc.) at 105° C. for 12 hr.

FT-IR spectra were obtained using a FT-IR spectrophotometer (Magna-IR560, Nicolet). The sample was cut into small pieces and characterized bya Fourier transform infrared spectrometer for evaluation of chemicalstructure.

Thermogravimetric analysis (TGA, TA instrument) was used to analyze thethermal properties of the sample. All analyses were performed withinaluminum pans under a dynamic nitrogen atmosphere between 25 and 600° C.The experiments were run at a scanning rate of 20° C./min and thenitrogen flow rate was 20 mL/min.

Results and Discussion

Formation of Composites

FIG. 4 shows films of the BC-soy resin (SPI) and GA treated BC-soy resin(SPI) composites. These materials were much softer and thicker than pureBC film. Their surfaces were smooth and thicknesses were uniform.

FIG. 5 shows the wet pellicle of BC-MFC and SEM images of its surfaceand cross section. The thickness of the wet BC-MFC pellicle was muchthicker than pure BC pellicle as MFC inserted into BC network. From SEMimages, MFC was observed to be present inside of MFC.

PEG not only was coated on the surface of BC pellicles but alsopenetrated into the BC fiber network (Cai, Z.; Kim, J. (2010). Bacterialcellulose/poly (ethylene glycol) composite: Characterization and firstevaluation of biocompatibility. Cellulose, 17, 83-91). The structuralmodification occurred as the water surrounding polyglucosan chains weredisplaced, including the formation of bonds between hydroxyl groups ofBC and PEG, and BC and PVA (Alberto, S.; Giovanni, T.; Anna, M. B.;Erinestina, D. P.; Elena, S.; Bruni, M. (2001). Characterization ofnative cellulose/poly(ethylene glycol) films. Macromolecular materialsand engineering, 286 (9), 524-538; Wang, J.; Gao, C.; Zhang, Y.; Wan, Y.(2010). Preparation and in vitro characterization of BC/PVA hydrogelcomposite for its potential use as artificial cornea biomaterial.Material science and engineering C, 30, 214-218).

Soy resin (SPI) and GA treated soy resin (SPI) also penetrated the BCnetwork and formed bonds between hydroxyl groups of BC and functionalgroups of soy resin (SPI).

Tensile Test of BC Based ‘Green’ Composites

Previous experiment indicated that the tensile properties of BC-soyresin (soy protein concentrate (SPC)) were much worse than BC-soy resin(SPI). Therefore, tensile testing for BC-soy resin (SPI) composite wascarried out in detail according to a modified ASTM D-882-02 standardtest method.

FIG. 6 presents the tensile test results for BC-soy resin (SPI)composites with different BC contents. The average tensile strength forpure SPI (0% BC) and pure BC (100% BC) were 9.61 MPa and 78.87 MParespectively. When BC was added into soy resins (SPI), the value oftensile strength of the BC-SPI increased dramatically compared with thatof pure soy resin (SPI). Tensile strength increased with increasing BCcontent. However, addition of BC decreased average tensile straincompared to that of soy resin (SPI). The average tensile strains forpure SPI and pure BC were 102.67% and 5.66%, respectively. As the BCcontent increased, the value of tensile strain for BC-soy resin (SPI)did not change significantly.

To further modify the tensile properties of BC-soy resin (SPI), MFC wasadded into the BC pellicle during culture, thereby producing BC-MFC-soyresin (SPI). Table 2 shows the tensile properties for BC, SPI, BC-soyresin (SPI), BC-MFC and BC-MFC-soy resin (SPI). It indicates that MFCaddition increases tensile strain properties of BC, but both the valuesof modulus and tensile strength decrease in BC-MFC composite. This waslikely due to inhomogeneous distribution of MFC in BC-MFC composite.After combining BC-MFC with soy resin (SPI), the tensile strength ofBC-MFC-SPI composite (1407.97 MPa) increased dramatically and was evenhigher than that of BC-SPI composite (1293.76 MPa). This was possiblybecause during preparation of the BC-MFC-SPI composite, SPI solutionreplaced water inside of the BC pellicle and re-arranged thedistribution of MFC, and made the composite homogenous and strong.

TABLE 2 Tensile properties of BC, SPI, BC-MFC and their based compositesBC- BC-SPI MFC-SPI Pure BC Pure SPI (SPI 50%) MFC-BC (SPI 50%) Young's2492.96 217.31 1293.76 589.95 1407.97 Modulus (240.33) (17.98) (19.55)(119.32) (272.98) (MPa) Tensile 78.87 9.61 51.02 33.17 49.47 Strength(10.76) (0.81) (1.48) (5.81) (11.53) (MPa) Tensile 5.66 102.67 6.22 8.376.82 Strain (1.01) (24.22) (1.26) (1.62) (1.28) (%)

GA crosslinking method was also employed in the study. Table 3 shows thetensile properties and moisture contents of GA-treated BC-soy resin(SPI) composites treated with different GA concentrations. As seen fromthe data in Table 3, the modulus values for GA treated BC-soy resin(SPI) increased dramatically from 1293.76 MPa to around 3600 MPa and thevalues of tensile strength and tensile strain decreased from 51.02 MPaand 6.22% to around 46 MPa and 1.40% compared to those of BC-soy resin(SPI) samples without GA treatment. The modulus, tensile strength andtensile stain showed little variation at concentrations of GA varyingfrom 2.5%-7.5%. However, all these values decreased significantly whenthe sample was treated with 10% GA.

GA treatment did not alter moisture content (MC) significantly and itsvalues for samples remained around 12%.

TABLE 3 Tensile properties and moisture content of GA treated BC-soyresin (SPI) composites GA Young's Tensile Tensile Moisture ConcentrationModulus Strength Strain Content (%) (MPa) (MPa) (%) (%)   0% GA 1293.7651.02 6.22 12.40 (19.55) (1.48) (1.26) (2.28) 2.5% GA 3625.17 46.72 1.4012.10 (772.74) (9.28) (0.46) (1.45) 5.0% GA 3604.27 44.61 1.40 12.50(427.37) (10.48) (0.32) (1.34) 7.5% GA 3644.97 46.15 1.41 12.80 (525.42)(11.67) (0.62) (1.67) 10.0% GA  2896.28 25.39 0.96 11.60 (658.26)(11.75) (0.31) (1.88)

FT-IR Spectra of BC Based ‘Green’ Composites

FIG. 7 depicts the FT-IR spectra of BC, BC-SPI, BC-MFC and BC-MFC-SPIsamples. Spectrum (a) is an FT-IR spectrum of pure BC. A band at 3345cm⁻¹ was owing to the presence of O—H stretching vibration, a band at2850 cm⁻¹ represented the aliphatic C—H stretching vibration, and a bandat 1020 cm⁻¹ represented was attributed to C—O—C stretching vibrations.Spectrum (c) shows FT-IR spectrum of pure BC-MFC composite. Theintensities and the width of peak at 3350 cm⁻¹ were larger than that ofBC, since BC-MFC has many hydroxyl groups. In spectra (b) and (d),strong peaks at 1600 cm⁻¹ indicated that BC-SPI and BC-MFC-SPI haveamino groups. Strong and broad peaks at 3340 cm⁻¹ indicated thatBC-MFC-SPI has many hydroxyl and carboxyl groups.

Thermo-Gravimetric Analysis (TGA) Testing of BC Based ‘Green’ Composites

Thermo-gravimetric analysis (TGA) can be used to characterize thermaldecomposition behavior. Test results of thermal stability anddecomposition of BC, BC-soy resin (SPI) are shown in FIGS. 8 a-b.

FIG. 8 a shows that pure BC remained stable up to 220° C., with 30%weight loss at 255° C., 50% weight loss at around 275° C. and almostcomplete weight loss at around 575° C.

FIG. 8 b shows that BC-soy resin (SPI) at a ratio of 1:1 remained stableup to 220° C., with 30% weight loss at 270° C., 50% weight loss ataround 310° C. and still only less than 80% weight loss at around 600°C.

The results indicated that BC-soy resin (SPI) composite had greaterthermal stability than pure BC.

Conclusion

This example demonstrates the production of films of BC based “green”composites, including BC-soy resin (SPI) composite, GA-treated soy resin(SPI) composite, BC-MFC composite and BC-MFC-soy resin (SPI). Thesecomposites had smooth surfaces and uniform thicknesses. SEM imagesindicated MFC penetrated into the network of BC nanofibers. Tensiletesting indicated that BC film could greatly increase the mechanicalproperties of soy resin (SPI) materials, including modulus and tensilestrength. MFC could further enhance this effect, yielding BC-MFC-soyresin (SPI) composites with higher moduli. Also, very high moduli forBC-soy resin (SPI) composites could be achieved by crosslinking using acrosslinking agent, GA. FTIR test showed the presence of amino andcarboxyl groups in BC-soy resin (SPI) and BC-MFC-soy resin (SPI)composites. TGA showed that BC-soy resin (SPI) composites had betterthermal stabilities than pure BC.

6.3 Example 3 Development of BC-Modified Sisal Fiber and its InterfacialShear Strength (IFSS) with Soy Protein

This example demonstrates the production of bacterial cellulose (BC)modified sisal fiber. The interfacial shear strength (IFSS) betweenBC-modified sisal fiber and soy protein isolate (SPI) resin was testedusing the microbond test. The results indicated that the interfacialadhesion between BC-modified sisal fiber and SPI resin was approximately18% higher than that between unmodified sisal fiber and SPI resin. Theimproved IFSS likely arises from the increased roughness associated withthe presence of nanoscale BC on the sisal fiber surface and thepotential for hydrogen bonding between the hydroxyl group present on theBC-modified fiber surface and functional groups in the SPI resin. Theexample also indicates that the IFSS of other fibers and resins cansimilarly be improved by BC surface modification.

Introduction

Natural fibers, such as sisal and ramie, have attracted much attentionin recent years for their function as reinforcement in many resins.These cellulose fibers are useful in forming environmentally friendlyand biodegradable composites with resins. However, the adhesion betweenfiber and resin can be weak (Kim, J. T.; Netravali, A. N. (2010). Effectof protein content in soy protein resins on their interfacial shearstrength with ramie fibers. Journal of adhesion science and technology,24, 203-215).

Bacterial cellulose (BC) is a promising biodegradable material withbroad potential for composite reinforcement. It is produced byAcetobacter xylinum, a Gram-negative, obligately aerobic bacterium, in anutritional fermentation medium at 30° C. for several days. BC has thesame chemical structure as other plant-based cellulose. However, BC iscomposed of fibers with diameters of only few nanometers and displaysmany desirable properties, including higher purity, highercrystallinity, higher degree of polymerization, higher tensile strength,higher modulus and stronger biological adaptability (Iguchi, M.;Yamanaka, S.; Budhiono, A. (2000) Bacterial cellulose—a masterpiece ofNature's arts. Journal of materials science, 35 (2), 261-270; Baeckdahl,H.; Helenius, G.; Bodin, A.; Nannmark, U.; Johansson, B. R.; Risberg,B.; Gatenholm, P. (2006). Mechanical properties of bacterial celluloseand interactions with smooth muscle cells. Biomaterials, 27 (9),2141-2149; Klemm, D.; Schumann, D.; Udhardt, U.; Marsch, S. (2001).Bacterial synthesized cellulose—artificial blood vessels formicrosurgery. Progress in polymer science, 26 (9), 1561-1603; Klemm, D.;Heublein, B.; Fink, H. P.; Bohn, Andreas. (2005). Cellulose: Fascinatingbiopolymer and sustainable raw material. Angewandte chemie,International edition, 44(22), 3358-3393; Fink, H. P.; Weigel, P.; Purz,H. J.; Ganster, J. (2001). Structure formation of regenerated cellulosematerials from NMMO-solutions. Progress in polymer science, 26(9),1473-1524).

Soy proteins are biodegradable polymers and have been used in ‘green’composites because of their worldwide availability and low price. Amongsoy proteins, soy protein isolate (SPI) is a highly refined or purifiedform with a minimum protein content of 90% on a moisture-free basis. Itis made from defatted soy flour, which has had most of the non-proteincomponents, fats and carbohydrates removed. SPI contains 18 differentamino acids that have polar functional groups such as hydroxyl, amineand carboxyl groups. These functional groups have a potential to reactor hydrogen bond to natural cellulose fibers that contain hydroxylgroups (Kim, J. T.; Netravali, A. N. (2010). Effect of protein contentin soy protein resins on their interfacial shear strength with ramiefibers. Journal of adhesion science and technology, 24, 203-215).

BC can be used to modify natural fibers surfaces, and improve theinterfacial adhesion between fiber and polymer resins for forming truly‘green’ fiber reinforced composites with enhanced properties and muchbetter durability (Pommet, M.; Juntaro, J.; Heng, J. Y. Y.; Mantalaris,A.; Lee, A. F.; Wilson, K.; Kalinka, G.; Shaffer, M. S. P.; Bismarck, A.(2008). Surface modification of natural fibers using bacteria:depositing bacterial cellulose onto natural fibers to createhierarchical fiber reinforced nanocomposites. Biomacromolecules, 9,1643-1651); Juntaro, J.; Pommet, M.; Kalinka, G; Mantalaris, A.;Shaffer, M. S. P.; Bismarck, Alexander. (2008). Creating hierarchicalstructures in renewable composites by attaching bacterial cellulose ontosisal fibers. Advanced materials, 20, 3122-3126).

Materials and Methods

Microorganism and Culture Media

Acetobacter xylinum, ATCC 23769, obtained from the American Type CultureCollection (ATCC, Manassas, Va.) was used as the model strain andmaintained on agar plates containing 25 g/L D-mannitol, 5 g/L yeastextract and 5 g/L tryptone and 20 g/L agar. The mannitol culture mediumused for BC production consisted of 25 g/L D-mannitol, 5 g/L yeastextract and 5 g/L tryptone.

Preparation of BC-Modified Sisal Fibers

The strain from the agar plate was inoculated into a conical flaskcontaining mannitol culture medium as the seed culture. The initial pHvalue of the medium was adjusted to 5.0 and was not regulated during theculture. The seed culture was incubated at 30° C. and 130 rpm on arotary shaker (MAXQ 4450, Thermo Scientific) for 2 days, and 9 mL ofthis was inoculated into a 150-mL sisal-mannitol culture medium (20sisal fibers, 12 cm length, around 0.1 g) in 1000-mL conical bottle forproduction of BC-sisal composite. The cultivation was carried out atinitial pH 5.0, 30° C. and 105 rpm in a rotary shaker for 3 days. Afterincubation, the surface of sisal fiber was coated a layer of BC. TheBC-modified sisal fiber was then harvested and washed successively withwater and 1% (w/v), aqueous NaOH at 90° C. for 15 min, and then washedby deionized water to remove all microbial product contaminants.

Preparation of SPI Resin

SPI resin was initially mixed with deionized water in a ratio of 1:10.The mixture was homogenized using a magnetic stirrer from 15 min afterwhich the mixture was maintained in a water bath at 75° C. for anadditional 30 min. This ‘precuring’ process helps denature the globularprotein by opening up the molecules. The pre-cured SPI resin was used tomake microbeads on a single sisal fiber.

Preparation of Sisal Fiber with SPI and of BC-Modified Sisal Fiber WithSPI Microbond Specimens

To evaluate the IFSS of sisal fiber-SPI and BC-modified sisal fiber-SPI,the art-known microbond technique was used. To prepare a microbondspecimen, a single sisal fiber or a single BC-modified sisal fiber wasmounted on a paper tab and glued at both ends using cyanoacrylate glue.Using the pre-cured SPI resin, a small microdrop (microbead) was placedon the sisal fiber or BC-modified sisal fiber. The fibers withmicrobeads were kept at room temperatures for at least 4 h beforeheating at 120° C. in an air-circulating oven for 60 min to cure theresin. This curing process has been shown to cross-link soy protein(Nam, S.; Netravali, A. N. (2006). Green composites. II.Environment-friendly, biodegradable composites using ramie fibers andsoy protein concentrate (SPC) resin. Fibers and polymer, 7, 380-388).All specimens used for the microbond tests were equilibrated at 21° C.and 65% relative humidity for 24 h prior to testing.

Interfacial Shear Strength (IFSS) Test

A schematic of the microbond test for IFSS is shown in FIG. 9. The fiberdiameter, d and embedded length, L, were measured prior to themicro-bond test using a calibrated optical microscope. To obtainaccurate measurements, d and L were measured again after the micro-bondtest. It should be noted that the microdrops tended to shrink indiameter and become smaller in diameter as the water evaporated, whilethere was no shrinkage in the lengthwise direction, as discussed later.The microbond test was performed on an Instron universal testingmachine, model 5566, with a microvise. The microvise plates were placedjust above the microbead and brought closer until they barely touchedthe fiber surface as shown in FIG. 9. The sisal fiber or BC-modifiedsisal fiber was then pulled out from the microbead at a crosshead speedof 0.2 mm/min until the microbead debonded. The interfacial shearstrength, τ, was calculated using the following equation:

${{IFSS}(\tau)} = \frac{F}{\pi \times d \times L}$

where F is the force required to debond the microbead. It was assumedthat the shear strength was uniform along the entire fiber/microbeadinterface. Twenty successful tests were conducted to obtain average IFSSvalues.

Results and Discussion

Production of BC-Modified Sisal Fibers

BC-modified sisal fibers were successfully produced in a rotary shakerafter cultivation. FIG. 10 shows the BC-modified sisal fibers where BCsurrounds the sisal fiber.

IFSS for Sisal Fiber with SPI and BC-Modified Sisal Fiber with SPI

FIG. 11 shows the comparison of IFSS values for sisal fiber with SPIresin and BC-modified sisal fiber with SPI resin. The IFSS values forsisal fiber with SPI and BC modified sisal with SPI were 3.033 MPa and3.575 MPa respectively. This indicated that IFSS tended to increase whenBC are coated on the surface of sisal fibers. The improved IFSS mightarise from the increase in roughness associated with the presence ofnanoscale bacterial cellulose on the sisal fiber surface and thepotential for hydrogen bonding between the hydroxyl group present on theBC-modified sisal fiber surface and functional groups in the SPI resin.

Conclusion

BC coated sisal fibers were produced. The interfacial shear strength(IFSS) between BC-modified sisal fiber and SPI resin was tested usingthe microbond test. The results indicated that BC-modified sisal fiberwith SPI resin had a much higher interfacial adhesion than that of sisalfiber with SPI resin. The improved IFSS may arise from the increase inroughness associated with the presence of nanoscale bacterial celluloseon the sisal fiber surface and the potential for hydrogen bondingbetween the hydroxyl group present on the BC-modified fiber surface andfunctional groups in the SPI resin.

6.4 Example 4 Development of BC-Based Membrane-Like ‘Green’ CompositesUsing Water-Soluble and Biodegradable Polymers

In this example, bacterial cellulose (BC)-based membrane-like ‘green’composites were produced by immersing wet BC pellicles in polyvinylalcohol (PVA) and polyethylene oxide (PEO) aqueous solutions. The BCcontent in BC-PVA and BC-PEO composites could be adjusted by usingdifferent concentrations of PVA and PEO aqueous solution. Thesecomposites had smooth surfaces and uniform thicknesses. SEM imagesindicated PVA and PEO not only penetrated into BC network, but alsofilled in pores among BC-nanofibers. Tensile test indicated that theinclusion of BC greatly increases the mechanical properties of PVA andPEO in composites, including modulus (stiffness) and tensile strength.Fourier transform infrared spectroscopy (FTIR) test showed that hydrogenbond could be formed between hydroxyl groups of BC and PEO. TGA showedthat BC-PVA and BC-PEO composites had much better thermal stabilitiesthan that of pure BC.

Introduction

During the past several decades, new advanced composites with excellentmechanical properties have been developed and used as metalreplacements. However, most composites are made using syntheticnon-degradable fibers, such as carbon, aramid and glass and polymers(resins), such as polyetheretherketone (PEEK) and epoxy. Thesecomposites pose a serious solid waste disposal problem owing todecreasing landfill space, widespread litter and pollution of marineenvironments (Chou and Frank, 1989; Mohanty, A. K.; Khan, Mubarak A.;Hinrichsen, G. (2000) Surface modification of jute and its influence onperformance of biodegradable jute—fabric/Biopol composites. Compositesscience and technology, 60 (7), 1115-1124). As a result, biodegradableand bio-based materials have attracted much attention in recent years.Very recently advanced ‘green’ composites were fabricated using soybased resins reinforced with liquid crystalline high strength cellulosefibers (Netravali, A. N.; Huang, X; Mizuta, K. (2007) Advanced ‘green’composite. Advanced composite materials, 16 (4), 269-282).

BC is produced by Acetobacter xylinum, a Gram-negative, obligatelyaerobic bacterium, in a nutritional fermentation medium as describedhereinabove. The medium at least contains carbon sources (mannitol,sucrose, fructose, etc.) and nitrogen sources (peptone, tryptone, yeastextract, etc.), and its optimum pH is 5.0. BC has the same chemicalstructure as other plant-based cellulose, has nanoscale fiber diametersand displays many unique properties including higher purity, highercrystallinity, higher degree of polymerization, higher tensile strength,higher modulus and stronger biological adaptability. Bacterial celluloseas a potential scaffold for tissue engineering of cartilage.Biomaterials, 26 (4), 419-431). However, the mechanical and thermalproperties of dry BC material are not ideal for some applications.

Water-soluble and biocompatible polymers were used in this example toenhance the properties of BC and form soft, uniform, and biocompatiblecomposites. Poly (vinyl alcohol) (PVA) and poly (ethylene oxide) (PEO)are two kinds of thermoplastic polymers that are also water-soluble,nonvolatile and biocompatible. PVA is also biodegradable. Hydrogen bondsare formed by hydroxyl groups of BC and PVA or PEO, thus furtherimproving the uniformity of films formed by the composite. A BC-PVAcomposite is known in the art (Wang, J.; Gao, C.; Zhang, Y.; Wan, Y.(2010). Preparation and in vitro characterization of BC/PVA hydrogelcomposite for its potential use as artificial cornea biomaterial.Material science and engineering C, 30, 214-218), but BC content in thecomposite was low and its mechanical properties were not ideal for someapplications.

BC-PEO or BC-PEG composites have been developed in the form of BC-PEOstrings during BC culture in PEO containing medium (Brown, E. E.;Laborie, M. G. (2007). Bioengineering bacterial cellulose/poly (ethyleneoxide) nanocomposites. Biomacromolecules, 8, 3074-3081) or in the formof BC-PEG film (Cai, Z.; Kim, J. (2010). Bacterial cellulose/poly(ethylene glycol) composite: Characterization and first evaluation ofbiocompatibility. Cellulose, 17, 83-91).

In this example, films of bacterial cellulose (BC)-based ‘green’composites were produced by immersing wet BC pellicles into PVA or PEOaqueous solutions. The composites had higher PVA or PEO content thanpreviously achieved and this content could be easily controlled asdesired, e.g., ratios of 1:3, 1:2, 1:1, 2:1; 3:1 etc., for ratios of PVAor PEO:BC. BC content in BC-PVA and BC-PEO composites were adjusted byusing different concentrations of PVA and PEO aqueous solution. Themechanical and structural properties of composites with BC-PVA andBC-PEO ratio were compared. These composites had smoother surfaces andmore uniform thicknesses, and their mechanical properties and thermalproperties were better than composites previously achieved in the art.

Materials and Methods

Microorganism and Culture Media

Acetobacter xylinum, ATCC 23769, obtained from the American Type CultureCollection (ATCC, Manassas, Va.) was used as the model strain andmaintained on agar plates containing 25 g/L D-mannitol, 5 g/L yeastextract and 5 g/L tryptone and 20 g/L agar. The mannitol culture mediumused for BC production consisted of 25 g/L D-mannitol, 5 g/L yeastextract and 5 g/L tryptone.

Preparation of BC Pellicles

The strain from the agar plate was inoculated into a conical flaskcontaining mannitol culture medium as the seed culture. The initial pHvalue of the medium was adjusted to 5.0 and was not regulated during theculture. The seed culture was incubated at 30° C. and 130 rpm on arotary shaker for 2 days, and 9 mL of this was inoculated into a 150-mLculture medium in 1000-mL conical bottle for production of BC. Thecultivation was carried out at initial pH 5.0 and 30° C. in a staticincubator for 10 days. After incubation, the BC pellicle produced on thesurface of mannitol culture medium was harvested and washed successivelywith water and 1% (w/v), aqueous NaOH at 90° C. for 15 min, and thenwashed by deionized water to remove all microbial product contaminants.

Preparation of Films of BC-PVA Composites

PVA powder (Aldrich) initially was added into the deionized water with aspecific percentage by weight, and the mixture was then stirred at 80°C. for 20 min to form PVA solution. The purified BC pellicle wasimmersed into the PVA solution in an 80° C. water bath for 2 hr, and wasthen allowed to remain in the PVA solution at room temperature for 12hr. The PVA containing BC pellicle was then transferred into deionizedwater for 30 min to remove superfluous PVA on the surface of BC pellicleand to stabilize the BC-PVA composite. The BC-PVA composite film wasdried at room temperature on a Teflon® plate for 48 hr until a constantweight was obtained. The BC-PVA composites with varying BC and PVAratios are shown in Table 4.

TABLE 4 BC and PVA ratio in BC-PVA composite Concentration of PVAsolution for BC BC and PVA ratio in pellicle treatment (w/v %) composite1.5% 3:1 2.0% 2:1 3.2% 1:1 4.0% 1:2 6.0% 1:3

Preparation of Films of BC-PEO Composites

PEO powder (Aldrich) was added into the deionized water at a desiredpercentage by weight (see Table 4). The mixture was stirred at 65° C.for 20 min to form PEO solution. The purified BC pellicle was immersedinto the PEO solution, and was then allowed to stay in the PEO solutionat room temperature for 12 hr. The PEO containing BC pellicle wastransferred into deionized water for 30 min to remove superfluous PEO onthe surface of BC pellicle and to stabilize the BC-PEO composite. TheBC-PEO composite film was dried at room temperature on a Teflon® platefor 24 hr until a constant weight was obtained. The BC-PEO compositeswith varying BC and PEO ratios are shown in Table 5.

TABLE 5 BC and PEO ratio in BC-PEO composite Concentration of PEOsolution for BC BC and PEO ratio in pellicle treatment (w/v %) composite1.3 2:1 2.0 1:1 3.0 1:2

Characterization of BC-PVA and BC-PEO Composites

Scanning electron microscopy (SEM) images were taken by a LEO 1550FESEM. Freeze dried samples of BC, BC-PVA and BC-PEO composites weresputter coated with gold and their morphologies were observed with theLEO 1550 FESEM at an accelerating voltage of 15 kV.

Tensile testing was performed by an Instron tensile test machine (model5566). The test specimens were prepared by cutting the membranes into 10mm wide and 60 mm long strips using a precise cutter. Young's moduli ofthe samples were determined from the tensile test results conductedaccording to ASTM D-882-02, using standard test methods for tensileelastic properties of thin plastic sheeting. Two ends of the specimenswere placed between the upper and lower jaws of the instrument, leavinga gauge length of 30 mm of the specimens between the two jaws. Strainrate of the instrument was 2%/min All samples were conditioned at 20° C.and 65% RH for 3 days prior to tensile test.

Moisture content (MC) was measured by a moisture/volatile tester (C.W.Brabender Instrument Inc.) at 105° C. for 12 hr. All samples wereconditioned at 21° C. and 65% RH for 3 days prior to MC testing.

FT-IR spectra were obtained using a FT-IR spectrophotometer (Magna-IR560, Nicolet). Samples were cut into small pieces and characterized by aFourier transform infrared spectrometer for the evaluation of chemicalstructures.

Thermogravimetric analysis (TGA, TA Instrument) was carried out usingstandard methods. TGA was used to analyze the thermal properties of thesample. All analyses were performed in aluminum pans under a dynamicnitrogen atmosphere between 25 and 600° C. for pure BC 25 and 800° C.for BC-PVA and BC-PEO composites. The experiments were run at a scanningrate of 20° C./min and the nitrogen flow rate was 20 mL/min.

Results and Discussion

Formation and SEM Images of BC-PVA and BC-PEO

FIG. 14 shows films of BC-PVA and BC-PEO composites. The materials aremuch softer than pure BC film. Their surfaces were smooth and theirthicknesses were uniform.

It has been reported previously that PEG may not only coat the surfaceof BC pellicles but may also penetrate into the BC fiber network (Cai,Z.; Kim, J. (2010). Bacterial cellulose/poly (ethylene glycol)composite: Characterization and first evaluation of biocompatibility.Cellulose, 17, 83-91). Furthermore, structural modification can occur asthe water surrounding polyglucosan chains are displaced, including theformation of bonds between hydroxyl groups of BC and PEG and of BC andPVA (Alberto, S.; Giovanni, T.; Anna, M. B.; Erinestina, D. P.; Elena,S.; Bruni, M. (2001). Characterization of native cellulose/poly(ethyleneglycol) films. Macromolecular materials and engineering, 286 (9),524-538; Wang, J.; Gao, C.; Zhang, Y.; Wan, Y. (2010). Preparation andin vitro characterization of BC/PVA hydrogel composite for its potentialuse as artificial cornea biomaterial. Material science and engineeringC, 30, 214-218).

FIGS. 15 a-f show SEM images of freeze dried pure BC, BC-PVA and BC-PEOcomposites. In FIGS. 15 a-b, the BC network and porous structure can beobserved clearly from the surface of the composite. The mean diameterBC-nanofibers is less than 100 nm and the diameter of pore ranges fromseveral dozens to several hundred nanometers. FIGS. 15 c-d showstructures of BC-PVA composites. FIGS. 15 e-f and show structures ofBC-PEO composites. PVA and PEO penetrated into the BC network structureand filled in most of pores of the BC. The diameter of BC-nanofibersbecame larger owing to the coverage of PVA and PEO. FIG. 15 e also showsthat many BC-nanofibers were embedded within the PVA and PEO layers,thus forming uniform composite structures.

Tensile Properties and Moisture Content of BC-PVA Composites

Tensile testing of BC-PVA composites was done according to modified ASTMD-882-02 standard test methods. Table 6 presents the tensile testresults for BC, PVA and BC-PVA composites at different ratios.

The mean modulus of BC was 2492.96 MPa while the mean modulus of PVA was12.77 MPa. Mean modulus values of BC-PVA composites were between thoseof BC and PVA. With the increasing of BC content, the mean modulus inBC-PVA composite increased as well. While the ratio of BC-PVA was 1:3,the mean modulus was only around 751.85 MPa. When the ratio increased to1:1, the mean modulus increased to 1590.12 MPa. It further increased to2250.75 MPa at the ratio of 3:1, which was very close to pure BC.

Mean tensile strength at the break of BC was 78.87 MPa and that for PVAwas approximately 5.36 MPa. The mean tensile strength values at breakfor BC-PVA composites were also between those between BC and PVA, andthe values increased when BC content in the composites increased.However, tensile strength differences between different BC-PVAcomposites were not so obvious compared to modulus. Tensile strengthranged from 29.54 MPa when BC:PVA ratio was 1:3 to 38.97 MPa when theratio was 3:1.

For tensile strain, the value of PVA was 234.25%, which was much higherthan BC's fracture strain of 5.66%. However, the tensile strains ofBC-PVA composites were lower than both those of BC and PVA. The tensilestrains ranged from 1.95% at the BC-PVA ratio of 3:1 to 5.39% at theratio of 1:3. This was likely owing to the two components in thecomposites having very different tensile strains.

For moisture content (MC), the value of BC was 17.80% while that of PVAwas only 3.85%. With increasing BC content in the BC-PVA composite, theMC increased as well.

TABLE 6 Tensile properties and moisture contents for BC, PVA and BC-PVAcomposites Tensile Modulus strength Tensile strain MC (MPa) (MPa) (%)(%) BC 2492.96 78.87 5.66 15.60 (240.33) (10.76) (1.01) PVA 12.77 5.36234.25 3.85 (4.63) (0.18) (33.62) BC:PVA 2250.75 38.97 1.95 12.87 (3:1)(133.06) (8.50) (0.35) BC:PVA 1685.89 33.47 2.21 9.77 (2:1) (71.83)(11.33) (0.69) BC:PVA 1590.12 32.56 2.38 7.67 (1:1) (190.12) (3.56)(0.62) BC:PVA 939.60 31.10 3.76 7.18 (1:2) (24.18) (1.56) (0.06) BC:PVA751.85 29.54 5.39 6.81 (1:3) (50.13) (6.05) (0.86)

BC-PEO Mechanical Properties

Tensile testing for BC-PEO composites was done according to modifiedASTM D-882-02 standard test methods. Table 7 presents the tensile testresults for BC, PEO and BC-PEO composite at different ratios.

The mean modulus of BC was 2492.96 MPa while the mean modulus of PEO was403.97 MPa. The mean modulus values of BC-PEO composites were betweenthose of BC and PEO. With the increasing of BC content, the mean modulusin BC-PEO composite increased as well. While the ratio of BC-PEO was1:2, the mean modulus was only around 1720.29 MPa. When the ratioincreased to 1:1, the mean modulus increased to 2027.99 MPa, and itwould further increased to 2275.10 MPa at the ratio of 2:1 which wasvery close to pure BC.

Mean tensile strength at break of BC was 78.87 MPa and that for PEO wasonly around 6.02 MPa. The mean tensile strength values at break forBC-PEO composites were also between those between BC and PEO, and thevalues increased when BC content in the composites increased.

For tensile strain, the value of PEO was 2.17%, which was much higherthan BC's 5.66%. However, the tensile strains of BC-PEO composites werelower than both those of BC and PEO. They ranged from 1.35% at theBC-PEO ratio of 2:1 to 2.04% at the ratio of 1:2. This was likely owingto the two components of the composites have very different tensilestrains.

As the melting point was lower than 105° C. for PEO and BC-PEOcomposites, moisture content for these composites was not tested.

TABLE 7 Tensile properties and moisture contents for BC, PEO and BC-PEOcomposites Tensile Modulus strength Tensile strain (MPa) (MPa) (%) BC2492.96 78.87 5.66 (240.33) (10.76) (0.72) PEO 403.97 6.02 2.17 (17.55)(0.48) (0.20) BC:PEO 2275.10 27.13 1.35 (2:1) (37.83) (7.58) (0.51)BC:PEO 2027.99 26.71 1.65 (1:1) (119.20) (9.56) (0.60) BC:PEO 1720.2926.05 2.04 (1:2) (100.36). (5.66) (0.53)

Tensile Properties Comparison Between BC-PVA and BC-PEO

Based on the results discussed above, BC pellicles can be used toincrease the mechanical properties of PVA and PEO materials. At the sameBC-polymer ratios, BC-PVA composites had higher modulus and tensilestrength while having lower tensile strains.

Fourier Transform Infrared Spectroscopy (FT-IR) of BC-PVA

FIG. 16 shows the Fourier transform infrared (FT-IR) spectra of BC,BC-PVA composites and PVA samples. Spectrum (a) is an FT-IR spectrum ofpure BC. A band at 3345 cm⁻¹ was owing to the presence of O—H stretchingvibration. A band at 2850 cm⁻¹ represented the aliphatic C—H stretchingvibration. A band at 1020 cm⁻¹ was attributed to C—O—C stretchingvibrations. Spectrum (e) is an FT-IR spectrum of pure PVA. Theintensities of bands at 3350 cm⁻¹, 2860 cm⁻¹ and 1070 cm⁻¹ are muchlarger than those of pure BC because the PVA have many hydroxyl groups,C—H bonds and C—O—C interactions respectively. Spectra (b), (c) and (d)show BC-PVA composites with different BC-PVA ratios. The intensities ofthe three bands are between those of pure BC and PVA and with increasingof BC content, the intensities of the three bands decreased. Thefrequencies of the C—O—C bands in BC-PVA composites also shifted alittle compared to those of BC and PVA which was owing to theintermolecular interacting by hydrogen bond between hydroxyl groups ofBC and PVA.

FT-IR of BC-PEO

FIG. 17 shows the FT-IR spectra of BC, BC-PEO composites and PEOsamples. Spectrum (a) shows FT-IR spectrum of pure BC. A band at 3345cm⁻¹ was owing to the presence of O—H stretching vibration. A band at2850 cm⁻¹ represented the aliphatic C—H stretching vibration. A band at1020 cm⁻¹ was attributed to C—O—C stretching vibrations. Spectrum eshows FT-IR spectrum of pure PEO. It had a strong band at 1450 cm⁻¹which represented CH₂ scissor vibration. The band at 3350 cm⁻¹ wasbroader than that of pure BC but the intensity was lower. Theintensities of bands at 2860 cm⁻¹ and 1070 cm⁻¹ were much larger thanthose of pure BC because the PEO have many C—H bonds and C—O—Cinteractions respectively. Spectra (b), (c) and (d) show BC-PEOcomposites with different BC-PEO ratios. The intensities of CH₂ scissorvibration was lower than PEO and the intensities of the O—H, C—H, C—O—Cbands are between those of pure BC and PEO and with increasing of BCcontent, the intensities of O—H bands increased and the intensities ofC—H, CH₂ and C—O—C bands decreased. The frequencies of the C—O—C bandsin BC-PEO composites also shifted a little compared to those of BC andPEO which was owing to the intermolecular interacting by hydrogen bondbetween hydroxyl groups of BC and PEO.

TGA of BC-PVA and BC-PEO Composites

Thermo-gravimetric analysis (TGA) provided information on thermaldecomposition behavior. Test results of thermal stability anddecomposition of BC, BC-PVA and BC-PEO are shown in FIGS. 18 a-c.

FIG. 18 a shows that pure BC remained stable up to 220° C., and 30%weight loss was at 255° C., 50% weight loss was at around 275° C. andalmost complete weight loss was at around 575° C.

FIG. 18 b shows that BC-PVA at a ratio of 1:1 remained stable up togreater than 260° C., with 30% weight loss at 315° C., 50% weight lossat around 340° C. and almost complete weight loss at greater than 800°C.

FIG. 18 c shows that BC-PEO at a ratio of 1:1 remained stable up togreater than 260° C., with 30% weight loss at 315° C., 50% weight lossat around 360° C. and almost complete weight loss at greater than 620°C.

The results indicated that BC-PVA and BC-PEO composites have greaterthermal stability than pure BC.

Conclusion

In this example, films of BC-PVA and BC-PEO composites with differingBC:PVA or BC:PEO ratios were produced by immersion methods. Thesecomposites had smooth surfaces and uniform thicknesses. SEM imagesindicated that PVA and PEO not only penetrated into the BC network, butalso filled in pores among BC nanofibers. Tensile testing indicated thatBC pellicles could greatly increase the mechanical properties of PVA andPEO materials, including increasing modulus and tensile strength. FTIRtesting showed that hydrogen bonds could form between hydroxyl groups ofBC and PEO. TGA showed that BC-PVA and BC-PEO had greater thermalstabilities than pure BC.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

1. A method for producing bacterial cellulose (BC) comprising: providing a bacterium wherein the bacterium is a bacterial cellulose-producing bacterium; providing a bacteria nutritional medium; culturing the bacterium in the bacteria nutritional medium under conditions to produce BC; and isolating BC produced by cultured bacteria from the bacteria nutritional medium, wherein: the bacteria nutritional medium comprises a carbon source, the carbon source is a plant-based seed extract, and the plant-based seed extract is derived from a plant-based seed comprising soluble sugars.
 2. The method of claim 1 wherein the bacteria is Acetobacter xylinum.
 3. The method of claim 1 wherein the seed is soy, wheat, corn or a legume.
 4. The method of claim 1 wherein the seed extract is soy flour extract (SFE).
 5. The method of claim 1 wherein the step of isolating BC comprises harvesting BC pellicles produced on the surface of the bacteria nutritional medium. 6-14. (canceled)
 15. The method of claim 1 wherein the bacteria nutritional medium comprises fibers that are introduced into the nutritional medium before or during the culturing step.
 16. The method of claim 1 comprising drying or hot-pressing the isolated BC, thereby forming a membrane. 17-20. (canceled)
 21. A composition comprising: bacterial cellulose (BC); and a soy-based resin.
 22. The composition of claim 21 that comprises 20 to 60% BC by weight.
 23. The composition of claim 21 wherein the composition is crosslinked.
 24. A composition comprising: bacterial cellulose (BC); and an agent selected from the group consisting of microfibrillated cellulose (MFC), nanofibrillated cellulose (NFC), cellulose nanowhisker, nanoparticle, nanoclay or nanocube, wherein the agent is interwoven or intercalated with the BC.
 25. The composition of claim 24 comprising a resin.
 26. (canceled)
 27. The composition of claim 25 wherein the resin is selected from the group consisting of biodegradable resin, water-soluble resin, natural resin, plant-based resin and non-toxic resin. 28-30. (canceled)
 31. The composition of claim 25 wherein the resin is a petroleum-based resin.
 32. The composition of claim 31 wherein the petroleum-based resin is an epoxy, vinyl, or unsaturated polyester-based resin.
 33. The composition of claim 25 wherein the resin is selected from the group consisting of polyethylene oxide (PEO), polyvinyl alcohol (PVA) and polyhydroxy alkanoate (PHA). 34-36. (canceled)
 37. The composition of claim 24 comprising fibers.
 38. (canceled)
 39. The composition of claim 37 wherein the fibers comprise a natural cellulose-based or protein-based material.
 40. The composition of claim 39 wherein the natural cellulose-based material is selected from the group consisting of cotton, linen, flax, sisal, ramie, hemp, kenaf, jute, bamboo, banana, pineapple, kapok, and combinations thereof.
 41. The composition of claim 39 wherein the natural protein-based material is selected from the group consisting of wool, silk, angora, cashmere, mohair, alpaca, milk protein, spider silk, and soy protein, and combinations thereof.
 42. The composition of claim 37 wherein the fibers comprise a polymeric material.
 43. The composition of claim 42 wherein the polymeric material is cellulose acetate, nylon, rayon, modacrylic, olefin, acrylic, polyester, polylactic acid, polylactic-co-glycolic acid (PLGA), polyurethane, aramid, or ultrahigh molecular weight polyethylene.
 44. The composition of claim 37 wherein the fibers comprise carbon or glass. 